Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework.

PubMed

Shewale, Jaiprakash G; Schneida, Elaine; Wilson, Jonathan; Walker, Jerilyn A; Batzer, Mark A; Sinha, Sudhir K

2007-03-01

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/microL of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.

A rapid and efficient DNA extraction protocol from fresh and frozen human blood samples.

PubMed

Guha, Pokhraj; Das, Avishek; Dutta, Somit; Chaudhuri, Tapas Kumar

2018-01-01

Different methods available for extraction of human genomic DNA suffer from one or more drawbacks including low yield, compromised quality, cost, time consumption, use of toxic organic solvents, and many more. Herein, we aimed to develop a method to extract DNA from 500 μL of fresh or frozen human blood. Five hundred microliters of fresh and frozen human blood samples were used for standardization of the extraction procedure. Absorbance at 260 and 280 nm, respectively, (A 260 /A 280 ) were estimated to check the quality and quantity of the extracted DNA sample. Qualitative assessment of the extracted DNA was checked by Polymerase Chain reaction and double digestion of the DNA sample. Our protocol resulted in average yield of 22±2.97 μg and 20.5±3.97 μg from 500 μL of fresh and frozen blood, respectively, which were comparable to many reference protocols and kits. Besides yielding bulk amount of DNA, our protocol is rapid, economical, and avoids toxic organic solvents such as Phenol. Due to unaffected quality, the DNA is suitable for downstream applications. The protocol may also be useful for pursuing basic molecular researches in laboratories having limited funds. © 2017 Wiley Periodicals, Inc.

Detection of human papillomavirus DNA in urine. A review of the literature.

PubMed

Vorsters, A; Micalessi, I; Bilcke, J; Ieven, M; Bogers, J; Van Damme, P

2012-05-01

The detection of human papillomavirus (HPV) DNA in urine, a specimen easily obtained by a non-invasive self-sampling method, has been the subject of a considerable number of studies. This review provides an overview of 41 published studies; assesses how different methods and settings may contribute to the sometimes contradictory outcomes; and discusses the potential relevance of using urine samples in vaccine trials, disease surveillance, epidemiological studies, and specific settings of cervical cancer screening. Urine sampling, storage conditions, sample preparation, DNA extraction, and DNA amplification may all have an important impact on HPV DNA detection and the form of viral DNA that is detected. Possible trends in HPV DNA prevalence in urine could be inferred from the presence of risk factors or the diagnosis of cervical lesions. HPV DNA detection in urine is feasible and may become a useful tool but necessitates further improvement and standardization.

Hybrid selection for sequencing pathogen genomes from clinical samples

PubMed Central

2011-01-01

We have adapted a solution hybrid selection protocol to enrich pathogen DNA in clinical samples dominated by human genetic material. Using mock mixtures of human and Plasmodium falciparum malaria parasite DNA as well as clinical samples from infected patients, we demonstrate an average of approximately 40-fold enrichment of parasite DNA after hybrid selection. This approach will enable efficient genome sequencing of pathogens from clinical samples, as well as sequencing of endosymbiotic organisms such as Wolbachia that live inside diverse metazoan phyla. PMID:21835008

A magnetic bead-based method for concentrating DNA from human urine for downstream detection.

PubMed

Bordelon, Hali; Russ, Patricia K; Wright, David W; Haselton, Frederick R

2013-01-01

Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×10(3) to 5×10(8) copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×10(6), 14×10(6), and 8×10(6) copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR.

A Magnetic Bead-Based Method for Concentrating DNA from Human Urine for Downstream Detection

PubMed Central

Bordelon, Hali; Russ, Patricia K.; Wright, David W.; Haselton, Frederick R.

2013-01-01

Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×103 to 5×108 copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×106, 14×106, and 8×106 copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR. PMID:23861895

Preferential access to genetic information from endogenous hominin ancient DNA and accurate quantitative SNP-typing via SPEX

PubMed Central

Brotherton, Paul; Sanchez, Juan J.; Cooper, Alan; Endicott, Phillip

2010-01-01

The analysis of targeted genetic loci from ancient, forensic and clinical samples is usually built upon polymerase chain reaction (PCR)-generated sequence data. However, many studies have shown that PCR amplification from poor-quality DNA templates can create sequence artefacts at significant levels. With hominin (human and other hominid) samples, the pervasive presence of highly PCR-amplifiable human DNA contaminants in the vast majority of samples can lead to the creation of recombinant hybrids and other non-authentic artefacts. The resulting PCR-generated sequences can then be difficult, if not impossible, to authenticate. In contrast, single primer extension (SPEX)-based approaches can genotype single nucleotide polymorphisms from ancient fragments of DNA as accurately as modern DNA. A single SPEX-type assay can amplify just one of the duplex DNA strands at target loci and generate a multi-fold depth-of-coverage, with non-authentic recombinant hybrids reduced to undetectable levels. Crucially, SPEX-type approaches can preferentially access genetic information from damaged and degraded endogenous ancient DNA templates over modern human DNA contaminants. The development of SPEX-type assays offers the potential for highly accurate, quantitative genotyping from ancient hominin samples. PMID:19864251

Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods.

PubMed

Greenspoon, S A; Sykes, K L V; Ban, J D; Pollard, A; Baisden, M; Farr, M; Graham, N; Collins, B L; Green, M M; Christenson, C C

2006-12-20

Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the software methods.

Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

PubMed Central

Carpenter, Meredith L.; Buenrostro, Jason D.; Valdiosera, Cristina; Schroeder, Hannes; Allentoft, Morten E.; Sikora, Martin; Rasmussen, Morten; Gravel, Simon; Guillén, Sonia; Nekhrizov, Georgi; Leshtakov, Krasimir; Dimitrova, Diana; Theodossiev, Nikola; Pettener, Davide; Luiselli, Donata; Sandoval, Karla; Moreno-Estrada, Andrés; Li, Yingrui; Wang, Jun; Gilbert, M. Thomas P.; Willerslev, Eske; Greenleaf, William J.; Bustamante, Carlos D.

2013-01-01

Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples. PMID:24568772

Long-term room temperature preservation of corpse soft tissue: an approach for tissue sample storage

PubMed Central

2011-01-01

Background Disaster victim identification (DVI) represents one of the most difficult challenges in forensic sciences, and subsequent DNA typing is essential. Collected samples for DNA-based human identification are usually stored at low temperature to halt the degradation processes of human remains. We have developed a simple and reliable procedure for soft tissue storage and preservation for DNA extraction. It ensures high quality DNA suitable for PCR-based DNA typing after at least 1 year of room temperature storage. Methods Fragments of human psoas muscle were exposed to three different environmental conditions for diverse time periods at room temperature. Storage conditions included: (a) a preserving medium consisting of solid sodium chloride (salt), (b) no additional substances and (c) garden soil. DNA was extracted with proteinase K/SDS followed by organic solvent treatment and concentration by centrifugal filter devices. Quantification was carried out by real-time PCR using commercial kits. Short tandem repeat (STR) typing profiles were analysed with 'expert software'. Results DNA quantities recovered from samples stored in salt were similar up to the complete storage time and underscored the effectiveness of the preservation method. It was possible to reliably and accurately type different genetic systems including autosomal STRs and mitochondrial and Y-chromosome haplogroups. Autosomal STR typing quality was evaluated by expert software, denoting high quality profiles from DNA samples obtained from corpse tissue stored in salt for up to 365 days. Conclusions The procedure proposed herein is a cost efficient alternative for storage of human remains in challenging environmental areas, such as mass disaster locations, mass graves and exhumations. This technique should be considered as an additional method for sample storage when preservation of DNA integrity is required for PCR-based DNA typing. PMID:21846338

Identification of human remains from the Second World War mass graves uncovered in Bosnia and Herzegovina

PubMed Central

Marjanović, Damir; Hadžić Metjahić, Negra; Čakar, Jasmina; Džehverović, Mirela; Dogan, Serkan; Ferić, Elma; Džijan, Snježana; Škaro, Vedrana; Projić, Petar; Madžar, Tomislav; Rod, Eduard; Primorac, Dragan

2015-01-01

Aim To present the results obtained in the identification of human remains from World War II found in two mass graves in Ljubuški, Bosnia and Herzegovina. Methods Samples from 10 skeletal remains were collected. Teeth and femoral fragments were collected from 9 skeletons and only a femoral fragment from 1 skeleton. DNA was isolated from bone and teeth samples using an optimized phenol/chloroform DNA extraction procedure. All samples required a pre-extraction decalcification with EDTA and additional post-extraction DNA purification using filter columns. Additionally, DNA from 12 reference samples (buccal swabs from potential living relatives) was extracted using the Qiagen DNA extraction method. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex ESI kit was used to simultaneously amplify 15 autosomal short tandem repeat (STR) loci, and PowerPlex Y23 was used to amplify 23 Y chromosomal STR loci. Matching probabilities were estimated using a standard statistical approach. Results A total of 10 samples were processed, 9 teeth and 1 femoral fragment. Nine of 10 samples were profiled using autosomal STR loci, which resulted in useful DNA profiles for 9 skeletal remains. A comparison of established victims' profiles against a reference sample database yielded 6 positive identifications. Conclusion DNA analysis may efficiently contribute to the identification of remains even seven decades after the end of the World War II. The significant percentage of positively identified remains (60%), even when the number of the examined possible living relatives was relatively small (only 12), proved the importance of cooperation with the members of the local community, who helped to identify the closest missing persons’ relatives and collect referent samples from them. PMID:26088850

Identification of human remains from the Second World War mass graves uncovered in Bosnia and Herzegovina.

PubMed

Marjanović, Damir; Hadžić Metjahić, Negra; Čakar, Jasmina; Džehverović, Mirela; Dogan, Serkan; Ferić, Elma; Džijan, Snježana; Škaro, Vedrana; Projić, Petar; Madžar, Tomislav; Rod, Eduard; Primorac, Dragan

2015-06-01

To present the results obtained in the identification of human remains from World War II found in two mass graves in Ljubuški, Bosnia and Herzegovina. Samples from 10 skeletal remains were collected. Teeth and femoral fragments were collected from 9 skeletons and only a femoral fragment from 1 skeleton. DNA was isolated from bone and teeth samples using an optimized phenol/chloroform DNA extraction procedure. All samples required a pre-extraction decalcification with EDTA and additional post-extraction DNA purification using filter columns. Additionally, DNA from 12 reference samples (buccal swabs from potential living relatives) was extracted using the Qiagen DNA extraction method. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex ESI kit was used to simultaneously amplify 15 autosomal short tandem repeat (STR) loci, and PowerPlex Y23 was used to amplify 23 Y chromosomal STR loci. Matching probabilities were estimated using a standard statistical approach. A total of 10 samples were processed, 9 teeth and 1 femoral fragment. Nine of 10 samples were profiled using autosomal STR loci, which resulted in useful DNA profiles for 9 skeletal remains. A comparison of established victims' profiles against a reference sample database yielded 6 positive identifications. DNA analysis may efficiently contribute to the identification of remains even seven decades after the end of the World War II. The significant percentage of positively identified remains (60%), even when the number of the examined possible living relatives was relatively small (only 12), proved the importance of cooperation with the members of the local community, who helped to identify the closest missing persons' relatives and collect referent samples from them.

BECon: a tool for interpreting DNA methylation findings from blood in the context of brain.

PubMed

Edgar, R D; Jones, M J; Meaney, M J; Turecki, G; Kobor, M S

2017-08-01

Tissue differences are one of the largest contributors to variability in the human DNA methylome. Despite the tissue-specific nature of DNA methylation, the inaccessibility of human brain samples necessitates the frequent use of surrogate tissues such as blood, in studies of associations between DNA methylation and brain function and health. Results from studies of surrogate tissues in humans are difficult to interpret in this context, as the connection between blood-brain DNA methylation is tenuous and not well-documented. Here, we aimed to provide a resource to the community to aid interpretation of blood-based DNA methylation results in the context of brain tissue. We used paired samples from 16 individuals from three brain regions and whole blood, run on the Illumina 450 K Human Methylation Array to quantify the concordance of DNA methylation between tissues. From these data, we have made available metrics on: the variability of cytosine-phosphate-guanine dinucleotides (CpGs) in our blood and brain samples, the concordance of CpGs between blood and brain, and estimations of how strongly a CpG is affected by cell composition in both blood and brain through the web application BECon (Blood-Brain Epigenetic Concordance; https://redgar598.shinyapps.io/BECon/). We anticipate that BECon will enable biological interpretation of blood-based human DNA methylation results, in the context of brain.

Production and certification of NIST Standard Reference Material 2372 Human DNA Quantitation Standard.

PubMed

Kline, Margaret C; Duewer, David L; Travis, John C; Smith, Melody V; Redman, Janette W; Vallone, Peter M; Decker, Amy E; Butler, John M

2009-06-01

Modern highly multiplexed short tandem repeat (STR) assays used by the forensic human-identity community require tight control of the initial amount of sample DNA amplified in the polymerase chain reaction (PCR) process. This, in turn, requires the ability to reproducibly measure the concentration of human DNA, [DNA], in a sample extract. Quantitative PCR (qPCR) techniques can determine the number of intact stretches of DNA of specified nucleotide sequence in an extremely small sample; however, these assays must be calibrated with DNA extracts of well-characterized and stable composition. By 2004, studies coordinated by or reported to the National Institute of Standards and Technology (NIST) indicated that a well-characterized, stable human DNA quantitation certified reference material (CRM) could help the forensic community reduce within- and among-laboratory quantitation variability. To ensure that the stability of such a quantitation standard can be monitored and that, if and when required, equivalent replacement materials can be prepared, a measurement of some stable quantity directly related to [DNA] is required. Using a long-established conventional relationship linking optical density (properly designated as decadic attenuance) at 260 nm with [DNA] in aqueous solution, NIST Standard Reference Material (SRM) 2372 Human DNA Quantitation Standard was issued in October 2007. This SRM consists of three quite different DNA extracts: a single-source male, a multiple-source female, and a mixture of male and female sources. All three SRM components have very similar optical densities, and thus very similar conventional [DNA]. The materials perform very similarly in several widely used gender-neutral assays, demonstrating that the combination of appropriate preparation methods and metrologically sound spectrophotometric measurements enables the preparation and certification of quantitation [DNA] standards that are both maintainable and of practical utility.

The effect of input DNA copy number on genotype call and characterising SNP markers in the humpback whale genome using a nanofluidic array.

PubMed

Bhat, Somanath; Polanowski, Andrea M; Double, Mike C; Jarman, Simon N; Emslie, Kerry R

2012-01-01

Recent advances in nanofluidic technologies have enabled the use of Integrated Fluidic Circuits (IFCs) for high-throughput Single Nucleotide Polymorphism (SNP) genotyping (GT). In this study, we implemented and validated a relatively low cost nanofluidic system for SNP-GT with and without Specific Target Amplification (STA). As proof of principle, we first validated the effect of input DNA copy number on genotype call rate using well characterised, digital PCR (dPCR) quantified human genomic DNA samples and then implemented the validated method to genotype 45 SNPs in the humpback whale, Megaptera novaeangliae, nuclear genome. When STA was not incorporated, for a homozygous human DNA sample, reaction chambers containing, on average 9 to 97 copies, showed 100% call rate and accuracy. Below 9 copies, the call rate decreased, and at one copy it was 40%. For a heterozygous human DNA sample, the call rate decreased from 100% to 21% when predicted copies per reaction chamber decreased from 38 copies to one copy. The tightness of genotype clusters on a scatter plot also decreased. In contrast, when the same samples were subjected to STA prior to genotyping a call rate and a call accuracy of 100% were achieved. Our results demonstrate that low input DNA copy number affects the quality of data generated, in particular for a heterozygous sample. Similar to human genomic DNA, a call rate and a call accuracy of 100% was achieved with whale genomic DNA samples following multiplex STA using either 15 or 45 SNP-GT assays. These calls were 100% concordant with their true genotypes determined by an independent method, suggesting that the nanofluidic system is a reliable platform for executing call rates with high accuracy and concordance in genomic sequences derived from biological tissue.

Human autosomal DNA and X chromosome STR profiles obtained from Chrysomya albiceps (Diptera: Calliphoridae) larvae used as a biological trace.

PubMed

Oliveira, T C; Santos, A B R; Rabelo, K C N; Souza, C A; Santos, S M; Crovella, S

2016-11-03

The use of insects to answer questions in criminal investigations, as well as a combination of forensic genetic techniques to obtain human DNA from the organisms, especially necrophagous dipterians, have gained ground in recent decades among researchers and professionals in this area. The objective of our study was to evaluate and compare two methods of human DNA extraction, commonly used for forensic samples, to obtain human autosomal DNA and X chromosome short tandem repeat profiles from the digestive tract of Chrysomya albiceps (Diptera: Calliphoridae) larvae. Immature specimens were collected from corpses at the Institute of Forensic Medicine of Pernambuco and raised in bovine ground meat to allow stabilization of the colony. Groups of larvae in the third instar were provided with bovine ground meat plus human blood for 48 h, dissected, and then subjected to DNA extraction. DNA was extracted using two methods: a DNA IQ™ kit and a phenol-chloroform method. Genomic DNA was amplified using AmpFℓSTR ® Identifiler ® Plus PCR and Argus-X-12 ® kits, and samples were sequenced to determine if the two extraction techniques generated reliable profiles that were compatible with a reference sample. The existence of comparable profiles from both techniques demonstrates the usefulness of dipteran larvae for obtaining human DNA from corpses, which can be further used to correlate genetic profiles in a crime scene when other traces are not available. However, several variables still require revision; thus, the technique should be further investigated for its validity, security, and, in particular, its reproducibility.

Comparing Ancient DNA Preservation in Petrous Bone and Tooth Cementum

PubMed Central

Margaryan, Ashot; Stenderup, Jesper; Lynnerup, Niels; Willerslev, Eske; Allentoft, Morten E.

2017-01-01

Large-scale genomic analyses of ancient human populations have become feasible partly due to refined sampling methods. The inner part of petrous bones and the cementum layer in teeth roots are currently recognized as the best substrates for such research. We present a comparative analysis of DNA preservation in these two substrates obtained from the same human skulls, across a range of different ages and preservation environments. Both substrates display significantly higher endogenous DNA content (average of 16.4% and 40.0% for teeth and petrous bones, respectively) than parietal skull bone (average of 2.2%). Despite sample-to-sample variation, petrous bone overall performs better than tooth cementum (p = 0.001). This difference, however, is driven largely by a cluster of viking skeletons from one particular locality, showing relatively poor molecular tooth preservation (<10% endogenous DNA). In the remaining skeletons there is no systematic difference between the two substrates. A crude preservation (good/bad) applied to each sample prior to DNA-extraction predicted the above/below 10% endogenous DNA threshold in 80% of the cases. Interestingly, we observe signficantly higher levels of cytosine to thymine deamination damage and lower proportions of mitochondrial/nuclear DNA in petrous bone compared to tooth cementum. Lastly, we show that petrous bones from ancient cremated individuals contain no measurable levels of authentic human DNA. Based on these findings we discuss the pros and cons of sampling the different elements. PMID:28129388

Thymidine kinase and mtDNA depletion in human cardiomyopathy: epigenetic and translational evidence for energy starvation

PubMed Central

Koczor, Christopher A.; Torres, Rebecca A.; Fields, Earl J.; Boyd, Amy; He, Stanley; Patel, Nilamkumar; Lee, Eva K.; Samarel, Allen M.

2013-01-01

This study addresses how depletion of human cardiac left ventricle (LV) mitochondrial DNA (mtDNA) and epigenetic nuclear DNA methylation promote cardiac dysfunction in human dilated cardiomyopathy (DCM) through regulation of pyrimidine nucleotide kinases. Samples of DCM LV and right ventricle (n = 18) were obtained fresh at heart transplant surgery. Parallel samples from nonfailing (NF) controls (n = 12) were from donor hearts found unsuitable for clinical use. We analyzed abundance of mtDNA and nuclear DNA (nDNA) using qPCR. LV mtDNA was depleted in DCM (50%, P < 0.05 each) compared with NF. No detectable change in RV mtDNA abundance occurred. DNA methylation and gene expression were determined using microarray analysis (GEO accession number: GSE43435). Fifty-seven gene promoters exhibited DNA hypermethylation or hypomethylation in DCM LVs. Among those, cytosolic thymidine kinase 1 (TK1) was hypermethylated. Expression arrays revealed decreased abundance of the TK1 mRNA transcript with no change in transcripts for other relevant thymidine metabolism enzymes. Quantitative immunoblots confirmed decreased TK1 polypeptide steady state abundance. TK1 activity remained unchanged in DCM samples while mitochondrial thymidine kinase (TK2) activity was significantly reduced. Compensatory TK activity was found in cardiac myocytes in the DCM LV. Diminished TK2 activity is mechanistically important to reduced mtDNA abundance and identified in DCM LV samples here. Epigenetic and genetic changes result in changes in mtDNA and in nucleotide substrates for mtDNA replication and underpin energy starvation in DCM. PMID:23695887

Developmental validation of the IrisPlex system: determination of blue and brown iris colour for forensic intelligence.

PubMed

Walsh, Susan; Lindenbergh, Alexander; Zuniga, Sofia B; Sijen, Titia; de Knijff, Peter; Kayser, Manfred; Ballantyne, Kaye N

2011-11-01

The IrisPlex system consists of a highly sensitive multiplex genotyping assay together with a statistical prediction model, providing users with the ability to predict blue and brown human eye colour from DNA samples with over 90% precision. This 'DNA intelligence' system is expected to aid police investigations by providing phenotypic information on unknown individuals when conventional DNA profiling is not informative. Falling within the new area of forensic DNA phenotyping, this paper describes the developmental validation of the IrisPlex assay following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines for the application of DNA-based eye colour prediction to forensic casework. The IrisPlex assay produces complete SNP genotypes with only 31pg of DNA, approximately six human diploid cell equivalents, and is therefore more sensitive than commercial STR kits currently used in forensics. Species testing revealed human and primate specificity for a complete SNP profile. The assay is capable of producing accurate results from simulated casework samples such as blood, semen, saliva, hair, and trace DNA samples, including extremely low quantity samples. Due to its design, it can also produce full profiles with highly degraded samples often found in forensic casework. Concordance testing between three independent laboratories displayed reproducible results of consistent levels on varying types of simulated casework samples. With such high levels of sensitivity, specificity, consistency and reliability, this genotyping assay, as a core part of the IrisPlex system, operates in accordance with SWGDAM guidelines. Furthermore, as we demonstrated previously, the IrisPlex eye colour prediction system provides reliable results without the need for knowledge on the bio-geographic ancestry of the sample donor. Hence, the IrisPlex system, with its model-based prediction probability estimation of blue and brown human eye colour, represents a useful tool for immediate application in accredited forensic laboratories, to be used for forensic intelligence in tracing unknown individuals from crime scene samples. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Extraction of DNA from human embryos after long-term preservation in formalin and Bouin's solutions.

PubMed

Nagai, Momoko; Minegishi, Katsura; Komada, Munekazu; Tsuchiya, Maiko; Kameda, Tomomi; Yamada, Shigehito

2016-05-01

The "Kyoto Collection of Human Embryos" at Kyoto University was begun in 1961. Although morphological analyses of samples in the Kyoto Collection have been performed, these embryos have been considered difficult to genetically analyze because they have been preserved in formalin or Bouin's solution for 20-50 years. Owing to the recent advances in molecular biology, it has become possible to extract DNA from long-term fixed tissues. The purpose of this study was to extract DNA from wet preparations of human embryo samples after long-term preservation in fixing solution. We optimized the DNA extraction protocol to be suitable for tissues that have been damaged by long-term fixation, including DNA-protein crosslinking damage. Diluting Li2 CO3 with 70% ethanol effectively removed picric acid from samples fixed in Bouin's solution. Additionally, 20.0 mg/mL proteinase was valuable to lyse the long-term fixed samples. The extracted DNA was checked with PCR amplification using several sets of primers and sequence analysis. The PCR products included at least 295- and 838-bp amplicons. These results show that the extracted DNA is applicable for genetic analyses, and indicate that old embryos in the Kyoto Collection should be made available for future studies. The protocol described in this study can successfully extract DNA from old specimens and, with improvements, should be applicable in research aiming to understand the molecular mechanisms of human congenital anomalies. © 2015 Japanese Teratology Society.

Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA.

PubMed

Pineda, Gina M; Montgomery, Anne H; Thompson, Robyn; Indest, Brooke; Carroll, Marion; Sinha, Sudhir K

2014-11-01

There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition. However, a system which can also assess the extent of degradation in a forensic sample will be a useful tool for DNA analysts. Possessing this information prior to genotyping will allow an analyst to more informatively make downstream decisions for the successful typing of a forensic sample without unnecessarily consuming DNA extract. Real-time PCR provides a reliable method for determining the amount and quality of amplifiable DNA in a biological sample. Alu are Short Interspersed Elements (SINE), approximately 300bp insertions which are distributed throughout the human genome in large copy number. The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human genome, which minimizes the individual specific variation possible when using a multi-copy target quantification system. This study utilizes two independent retrotransposon genomic targets to obtain quantification of an 80bp "short" DNA fragment and a 207bp "long" DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™. The ratio of the two quantitation values provides a "Degradation Index", or a qualitative measure of a sample's extent of degradation. The Degradation Index was found to be predictive of the observed loss of STR markers and alleles as degradation increases. Use of a synthetic target as an internal positive control (IPC) provides an additional assessment for the presence of PCR inhibitors in the test sample. In conclusion, a DNA based qualitative/quantitative/inhibition assessment system that accurately predicts the status of a biological sample, will be a valuable tool for deciding which DNA test kit to utilize and how much target DNA to use, when processing compromised forensic samples for DNA testing. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

An accurate bacterial DNA quantification assay for HTS library preparation of human biological samples.

PubMed

Seashols-Williams, Sarah; Green, Raquel; Wohlfahrt, Denise; Brand, Angela; Tan-Torres, Antonio Limjuco; Nogales, Francy; Brooks, J Paul; Singh, Baneshwar

2018-05-17

Sequencing and classification of microbial taxa within forensically relevant biological fluids has the potential for applications in the forensic science and biomedical fields. The quantity of bacterial DNA from human samples is currently estimated based on quantity of total DNA isolated. This method can miscalculate bacterial DNA quantity due to the mixed nature of the sample, and consequently library preparation is often unreliable. We developed an assay that can accurately and specifically quantify bacterial DNA within a mixed sample for reliable 16S ribosomal DNA (16S rDNA) library preparation and high throughput sequencing (HTS). A qPCR method was optimized using universal 16S rDNA primers, and a commercially available bacterial community DNA standard was used to develop a precise standard curve. Following qPCR optimization, 16S rDNA libraries from saliva, vaginal and menstrual secretions, urine, and fecal matter were amplified and evaluated at various DNA concentrations; successful HTS data were generated with as low as 20 pg of bacterial DNA. Changes in bacterial DNA quantity did not impact observed relative abundances of major bacterial taxa, but relative abundance changes of minor taxa were observed. Accurate quantification of microbial DNA resulted in consistent, successful library preparations for HTS analysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development.

PubMed

Vera-Rodriguez, M; Diez-Juan, A; Jimenez-Almazan, J; Martinez, S; Navarro, R; Peinado, V; Mercader, A; Meseguer, M; Blesa, D; Moreno, I; Valbuena, D; Rubio, C; Simon, C

2018-04-01

What is the origin and composition of cell-free DNA in human embryo spent culture media? Cell-free DNA from human embryo spent culture media represents a mix of maternal and embryonic DNA, and the mixture can be more complex for mosaic embryos. In 2016, ~300 000 human embryos were chromosomally and/or genetically analyzed using preimplantation genetic testing for aneuploidies (PGT-A) or monogenic disorders (PGT-M) before transfer into the uterus. While progress in genetic techniques has enabled analysis of the full karyotype in a single cell with high sensitivity and specificity, these approaches still require an embryo biopsy. Thus, non-invasive techniques are sought as an alternative. This study was based on a total of 113 human embryos undergoing trophectoderm biopsy as part of PGT-A analysis. For each embryo, the spent culture media used between Day 3 and Day 5 of development were collected for cell-free DNA analysis. In addition to the 113 spent culture media samples, 28 media drops without embryo contact were cultured in parallel under the same conditions to use as controls. In total, 141 media samples were collected and divided into two groups: one for direct DNA quantification (53 spent culture media and 17 controls), the other for whole-genome amplification (60 spent culture media and 11 controls) and subsequent quantification. Some samples with amplified DNA (N = 56) were used for aneuploidy testing by next-generation sequencing; of those, 35 samples underwent single-nucleotide polymorphism (SNP) sequencing to detect maternal contamination. Finally, from the 35 spent culture media analyzed by SNP sequencing, 12 whole blastocysts were analyzed by fluorescence in situ hybridization (FISH) to determine the level of mosaicism in each embryo, as a possible origin for discordance between sample types. Trophectoderm biopsies and culture media samples (20 μl) underwent whole-genome amplification, then libraries were generated and sequenced for an aneuploidy study. For SNP sequencing, triads including trophectoderm DNA, cell-free DNA, and follicular fluid DNA were analyzed. In total, 124 SNPs were included with 90 SNPs distributed among all autosomes and 34 SNPs located on chromosome Y. Finally, 12 whole blastocysts were fixed and individual cells were analyzed by FISH using telomeric/centromeric probes for the affected chromosomes. We found a higher quantity of cell-free DNA in spent culture media co-cultured with embryos versus control media samples (P ≤ 0.001). The presence of cell-free DNA in the spent culture media enabled a chromosomal diagnosis, although results differed from those of trophectoderm biopsy analysis in most cases (67%). Discordant results were mainly attributable to a high percentage of maternal DNA in the spent culture media, with a median percentage of embryonic DNA estimated at 8%. Finally, from the discordant cases, 91.7% of whole blastocysts analyzed by FISH were mosaic and 75% of the analyzed chromosomes were concordant with the trophectoderm DNA diagnosis instead of the cell-free DNA result. This study was limited by the sample size and the number of cells analyzed by FISH. This is the first study to combine chromosomal analysis of cell-free DNA, SNP sequencing to identify maternal contamination, and whole-blastocyst analysis for detecting mosaicism. Our results provide a better understanding of the origin of cell-free DNA in spent culture media, offering an important step toward developing future non-invasive karyotyping that must rely on the specific identification of DNA released from human embryos. This work was funded by Igenomix S.L. There are no competing interests.

Comparative Study of Seven Commercial Kits for Human DNA Extraction from Urine Samples Suitable for DNA Biomarker-Based Public Health Studies

PubMed Central

El Bali, Latifa; Diman, Aurélie; Bernard, Alfred; Roosens, Nancy H. C.; De Keersmaecker, Sigrid C. J.

2014-01-01

Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at −20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies. PMID:25365790

Genetic identification of missing persons: DNA analysis of human remains and compromised samples.

PubMed

Alvarez-Cubero, M J; Saiz, M; Martinez-Gonzalez, L J; Alvarez, J C; Eisenberg, A J; Budowle, B; Lorente, J A

2012-01-01

Human identification has made great strides over the past 2 decades due to the advent of DNA typing. Forensic DNA typing provides genetic data from a variety of materials and individuals, and is applied to many important issues that confront society. Part of the success of DNA typing is the generation of DNA databases to help identify missing persons and to develop investigative leads to assist law enforcement. DNA databases house DNA profiles from convicted felons (and in some jurisdictions arrestees), forensic evidence, human remains, and direct and family reference samples of missing persons. These databases are essential tools, which are becoming quite large (for example the US Database contains 10 million profiles). The scientific, governmental and private communities continue to work together to standardize genetic markers for more effective worldwide data sharing, to develop and validate robust DNA typing kits that contain the reagents necessary to type core identity genetic markers, to develop technologies that facilitate a number of analytical processes and to develop policies to make human identity testing more effective. Indeed, DNA typing is integral to resolving a number of serious criminal and civil concerns, such as solving missing person cases and identifying victims of mass disasters and children who may have been victims of human trafficking, and provides information for historical studies. As more refined capabilities are still required, novel approaches are being sought, such as genetic testing by next-generation sequencing, mass spectrometry, chip arrays and pyrosequencing. Single nucleotide polymorphisms offer the potential to analyze severely compromised biological samples, to determine the facial phenotype of decomposed human remains and to predict the bioancestry of individuals, a new focus in analyzing this type of markers. Copyright © 2012 S. Karger AG, Basel.

Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus-associated invasive carcinoma.

PubMed

Jeannot, Emmanuelle; Becette, Véronique; Campitelli, Maura; Calméjane, Marie-Ange; Lappartient, Emmanuelle; Ruff, Evelyne; Saada, Stéphanie; Holmes, Allyson; Bellet, Dominique; Sastre-Garau, Xavier

2016-10-01

Specific human papillomavirus genotypes are associated with most ano-genital carcinomas and a large subset of oro-pharyngeal carcinomas. Human papillomavirus DNA is thus a tumour marker that can be detected in the blood of patients for clinical monitoring. However, data concerning circulating human papillomavirus DNA in cervical cancer patients has provided little clinical value, due to insufficient sensitivity of the assays used for the detection of small sized tumours. Here we took advantage of the sensitive droplet digital PCR method to identify circulating human papillomavirus DNA in patients with human papillomavirus-associated carcinomas. A series of 70 serum specimens, taken at the time of diagnosis, between 2002 and 2013, were retrospectively analyzed in patients with human papillomavirus-16 or human papillomavirus-18-associated carcinomas, composed of 47 cases from the uterine cervix, 15 from the anal canal and 8 from the oro-pharynx. As negative controls, 18 serum samples from women with human papillomavirus-16-associated high-grade cervical intraepithelial neoplasia were also analyzed. Serum samples were stored at -80°C (27 cases) or at -20°C (43 cases). DNA was isolated from 200 µl of serum or plasma and droplet digital PCR was performed using human papillomavirus-16 E7 and human papillomavirus-18 E7 specific primers. Circulating human papillomavirus DNA was detected in 61/70 (87%) serum samples from patients with carcinoma and in no serum from patients with cervical intraepithelial neoplasia. The positivity rate increased to 93% when using only serum stored at -80°C. Importantly, the two patients with microinvasive carcinomas in this series were positive. Quantitative evaluation showed that circulating viral DNA levels in cervical cancer patients were related to the clinical stage and tumour size, ranging from 55 ± 85 copies/ml (stage I) to 1774 ± 3676 copies/ml (stage IV). Circulating human papillomavirus DNA is present in patients with human papillomavirus-associated invasive cancers even at sub-clinical stages and its level is related to tumour dynamics. Droplet digital PCR is a promising method for circulating human papillomavirus DNA detection and quantification. No positivity was found in patients with human papillomavirus-associated high grade cervical intraepithelial neoplasia.

Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus‐associated invasive carcinoma

PubMed Central

Jeannot, Emmanuelle; Becette, Véronique; Campitelli, Maura; Calméjane, Marie‐Ange; Lappartient, Emmanuelle; Ruff, Evelyne; Saada, Stéphanie; Holmes, Allyson; Bellet, Dominique

2016-01-01

Abstract Specific human papillomavirus genotypes are associated with most ano‐genital carcinomas and a large subset of oro‐pharyngeal carcinomas. Human papillomavirus DNA is thus a tumour marker that can be detected in the blood of patients for clinical monitoring. However, data concerning circulating human papillomavirus DNA in cervical cancer patients has provided little clinical value, due to insufficient sensitivity of the assays used for the detection of small sized tumours. Here we took advantage of the sensitive droplet digital PCR method to identify circulating human papillomavirus DNA in patients with human papillomavirus‐associated carcinomas. A series of 70 serum specimens, taken at the time of diagnosis, between 2002 and 2013, were retrospectively analyzed in patients with human papillomavirus‐16 or human papillomavirus‐18‐associated carcinomas, composed of 47 cases from the uterine cervix, 15 from the anal canal and 8 from the oro‐pharynx. As negative controls, 18 serum samples from women with human papillomavirus‐16‐associated high‐grade cervical intraepithelial neoplasia were also analyzed. Serum samples were stored at −80°C (27 cases) or at −20°C (43 cases). DNA was isolated from 200 µl of serum or plasma and droplet digital PCR was performed using human papillomavirus‐16 E7 and human papillomavirus‐18 E7 specific primers. Circulating human papillomavirus DNA was detected in 61/70 (87%) serum samples from patients with carcinoma and in no serum from patients with cervical intraepithelial neoplasia. The positivity rate increased to 93% when using only serum stored at −80°C. Importantly, the two patients with microinvasive carcinomas in this series were positive. Quantitative evaluation showed that circulating viral DNA levels in cervical cancer patients were related to the clinical stage and tumour size, ranging from 55 ± 85 copies/ml (stage I) to 1774 ± 3676 copies/ml (stage IV). Circulating human papillomavirus DNA is present in patients with human papillomavirus‐associated invasive cancers even at sub‐clinical stages and its level is related to tumour dynamics. Droplet digital PCR is a promising method for circulating human papillomavirus DNA detection and quantification. No positivity was found in patients with human papillomavirus‐associated high grade cervical intraepithelial neoplasia. PMID:27917295

Estimation and quantification of human DNA in dental calculus: A pilot study.

PubMed

Singh, Udita; Goel, Saurabh

2017-01-01

Identification using DNA has proved its accuracy multiple times in the field of forensic investigations. Investigators usually rely on either teeth or bone as the DNA reservoirs. However, there are instances where the skeletal or dental remains are not available or not preserved properly. Moreover, due to religious beliefs, the family members of the dead do not allow the investigating team to damage the remains for the sole purpose of identification. To investigate the presence of human DNA in dental calculus and to quantify the amount, if present. This prospective single-blinded pilot study included twenty subjects selected from the patients visiting a dental college. The samples of dental calculus were collected from the thickest portion of calculus deposited on the lingual surfaces of mandibular incisors. These samples were decontaminated and subjected to gel electrophoresis for DNA extraction. DNA was found in 85% cases. The amount of DNA varied from 21 to 37 μg/ml of dental calculus. Dental calculus is a rich reservoir of human DNA.

Design, optimisation and preliminary validation of a human specific loop-mediated amplification assay for the rapid detection of human DNA at forensic crime scenes.

PubMed

Hird, H J; Brown, M K

2017-11-01

The identification of samples at a crime scene which require forensic DNA typing has been the focus of recent research interest. We propose a simple, but sensitive analysis system which can be deployed at a crime scene to identify crime scene stains as human or non-human. The proposed system uses the isothermal amplification of DNA in a rapid assay format, which returns results in as little as 30min from sampling. The assay system runs on the Genie II device, a proven in-field detection system which could be deployed at a crime scene. The results presented here demonstrate that the system was sufficiently specific and sensitive and was able to detect the presence of human blood, semen and saliva on mock forensic samples. Copyright © 2017. Published by Elsevier B.V.

Biochemical quantification of DNA in human articular and septal cartilage using PicoGreen and Hoechst 33258.

PubMed

McGowan, K B; Kurtis, M S; Lottman, L M; Watson, D; Sah, R L

2002-07-01

To compare two fluorometric assays, utilizing (1) the bisbenzimidazole Hoechst 33258 and (2) PicoGreen, for determining DNA content in human cartilage. Human articular and nasal septal cartilage explants were digested using proteinase K. Portions of sample digest were analysed for intrinsic and dye-enhanced fluorescence with either Hoechst 33258 or PicoGreen. Intrinsic tissue fluorescence in both articular and septal cartilage increased with age and was prominent at wavelengths used for Hoechst 33258 but relatively low at wavelengths used for PicoGreen. The relative contribution of intrinsic fluorescence to total dye-enhanced fluorescence of human cartilage was markedly greater for Hoechst 33258 (19-57%) than for PicoGreen (2-7%). Thus, in many situations, DNA in human cartilage can be assayed using PicoGreen without the need to correct for intrinsic cartilage fluorescence. The enhancement of fluorescence by each dye was found to be specific for DNA, as shown by fluorescence spectra, >90% sensitivity to DNase, and resistance to RNase. In addition, little or no interference was caused by non-DNA tissue components, since DNA caused an equal enhancement in the absence or presence of proteinase K digested human cartilage, once intrinsic cartilage fluorescence was subtracted. PicoGreen was more sensitive for assaying DNA (0.9ng DNA/ml) than Hoechst 33258 (6ng DNA/ml) and can also be used in a microplate reader. PicoGreen can be used in a rapid and sensitive assay to quantify DNA in small samples of human cartilage. Copyright 2002 Published by Elsevier Science Ltd on behalf of OsteoArthritis Research Society International.

Real-time PCR detection and quantification of nine potential sources of fecal contamination by analysis of mitochondrial Cytochrome b targets

USGS Publications Warehouse

Schill, W.B.; Mathes, M.V.

2008-01-01

We designed and tested real-time PCR probe/primer sets to detect and quantify Cytochrome b sequences of mitochondrial DNA (mtDNA) from nine vertebrate species of pet (dog), farm (cow, chicken, sheep, horse, pig), wildlife (Canada goose, white-tailed deer), and human. Linear ranges of the assays were from 101 to 108 copies/??l. To formally test the performance of the assays, twenty blinded fecal suspension samples were analyzed by real-time PCR to identify the source of the feces. Sixteen of the twenty samples were correctly and unambiguously identified. Average sensitivity was calculated to be 0.850, while average specificity was found to be 0.994. One beef cow sample was not detected, but mtDNA from 11 other beef cattle of both sexes and varying physiological states was found in concentrations similar (3.45 ?? 107 copies/g) to thatfound in human feces (1.1 ?? 107 copies/g). Thus, environmental conditions and sample handling are probably important factors for successful detection of fecal mtDNA. When sewage samples were analyzed, only human mtDNA (7.2 ?? 104 copies/100 mL) was detected. With a detection threshold of 250 copies/reaction, an efficient concentration and purification method resulted in a final detection limit for human feces of 1.8 mg/100 mL water.

Immunological responses against human papilloma virus and human papilloma virus induced laryngeal cancer.

PubMed

Chitose, Shun-ichi; Sakazaki, T; Ono, T; Kurita, T; Mihashi, H; Nakashima, T

2010-06-01

This study aimed to clarify the local immune status in the larynx in the presence of infection or carcinogenesis associated with human papilloma virus. Cytological samples (for human papilloma virus detection) and laryngeal secretions (for immunoglobulin assessment) were obtained from 31 patients with laryngeal disease, during microscopic laryngeal surgery. On histological examination, 12 patients had squamous cell carcinoma, four had laryngeal papilloma and 15 had other benign laryngeal disease. Cytological samples were tested for human papilloma virus DNA using the Hybrid Capture 2 assay. High risk human papilloma virus DNA was detected in 25 per cent of patients (three of 12) with laryngeal cancer. Low risk human papilloma virus DNA was detected only in three laryngeal papilloma patients. The mean laryngeal secretion concentrations of immunoglobulins M, G and A and secretory immunoglobulin A in human papilloma virus DNA positive patients were more than twice those in human papilloma virus DNA negative patients. A statistically significant difference was observed between the secretory immunoglobulin A concentrations in the two groups. Patients with laryngeal cancer had higher laryngeal secretion concentrations of each immunoglobulin type, compared with patients with benign laryngeal disease. The study assessed the mean laryngeal secretion concentrations of each immunoglobulin type in the 12 laryngeal cancer patients, comparing human papilloma virus DNA positive patients (n = 3) and human papilloma virus DNA negative patients (n = 9); the mean concentrations of immunoglobulins M, G and A and secretory immunoglobulin A tended to be greater in human papilloma virus DNA positive cancer patients, compared with human papilloma virus DNA negative cancer patients. These results suggest that the local laryngeal immune response is activated by infection or carcinogenesis due to human papilloma virus. The findings strongly suggest that secretory IgA has inhibitory activity against infection or carcinogenesis associated with human papilloma virus in the larynx.

Molecular detection of Anaplasma phagocytophilum DNA in the lesser horseshoe bat (Rhinolophus hipposideros) guano.

PubMed

Afonso, E; Goydadin, A-C

2018-05-30

Although bats are increasingly recognised as potential reservoir hosts of human zoonotic pathogens, bacteria in bats are still poorly studied. To investigate the DNA faecal prevalence of the bacterium Anaplasma phagocytophilum, we sampled 23 lesser horseshoe bat (Rhinolophus hipposideros) maternity colonies located in buildings (churches, barns) in rural villages of eastern France. A total of 552 faecal samples were collected from 278 individuals. Anaplasma phagocytophilum DNA was detected in the faeces of 63 individuals (22.7%). Such high prevalence might suggest persistent infection in bats and/or a frequent consumption of insect preys carrying bacteria. Faecal DNA prevalence varied highly among colonies but was not related to the colony size. Faecal DNA prevalence was the highest in the Jura Department, where the density of ticks is known to be the highest across the study area. Because the sampled bats live in close proximity to humans, we discuss how concerning the presence of A. phagocytophilum DNA in bat guano is for humans frequenting places of worship that shelter bats. We also advocate future research to understand what a high faecal DNA prevalence in bat guano really implicates in terms of bacteria transmission.

A new human genetic resource: a DNA bank established as part of the Avon longitudinal study of pregnancy and childhood (ALSPAC).

PubMed

Jones, R W; Ring, S; Tyfield, L; Hamvas, R; Simmons, H; Pembrey, M; Golding, J

2000-09-01

We describe a unique human DNA resource forming part of the Avon Longitudinal Study of Pregnancy and Childhood (ALSPAC), a longitudinal cohort study involving 14 000 children and their families living in a geographically defined area of England. The DNA bank will underpin the search for associations between genetic polymorphisms and common health outcomes. The opportunities to collect blood samples suitable for DNA extraction are necessarily limited, and the samples themselves have often been treated in different ways and have varied storage histories. With the need to maximise yields, the choice of DNA extraction method is critical to the success of the bank and we have investigated the suitability of various commercial and in-house methods of DNA extraction. Various steps have been taken to minimise errors in sample address and identification, including the use of a pipetting robot for dilution and transfer of samples between 96-well arrays to provide aliquots suitable for PCR. The robot has been programmed to cope with concentrated viscous DNA solutions.

Is MMTV associated with human breast cancer? Maybe, but probably not.

PubMed

Perzova, Raisa; Abbott, Lynn; Benz, Patricia; Landas, Steve; Khan, Seema; Glaser, Jordan; Cunningham, Coleen K; Poiesz, Bernard

2017-10-13

Conflicting results regarding the association of MMTV with human breast cancer have been reported. Published sequence data have indicated unique MMTV strains in some human samples. However, concerns regarding contamination as a cause of false positive results have persisted. We performed PCR assays for MMTV on human breast cancer cell lines and fresh frozen and formalin fixed normal and malignant human breast epithelial samples. Assays were also performed on peripheral blood mononuclear cells from volunteer blood donors and subjects at risk for human retroviral infections. In addition, assays were performed on DNA samples from wild and laboratory mice. Sequencing of MMTV positive samples from both humans and mice were performed and phylogenetically compared. Using PCR under rigorous conditions to prevent and detect "carryover" contamination, we did detect MMTV DNA in human samples, including breast cancer. However, the results were not consistent and seemed to be an artifact. Further, experiments indicated that the probable source of false positives was murine DNA, containing endogenous MMTV, present in our building. However, comparison of published and, herein, newly described MMTV sequences with published data, indicates that there are some very unique human MMTV sequences in the literature. While we could not confirm the true presence of MMTV in our human breast cancer subjects, the data indicate that further, perhaps more traditional, retroviral studies are warranted to ascertain whether MMTV might rarely be the cause of human breast cancer.

Human Endometrial DNA Methylome Is Cycle-Dependent and Is Associated With Gene Expression Regulation

PubMed Central

Houshdaran, Sahar; Zelenko, Zara; Irwin, Juan C.

2014-01-01

Human endometrium undergoes major gene expression changes, resulting in altered cellular functions in response to cyclic variations in circulating estradiol and progesterone, largely mediated by transcription factors and nuclear receptors. In addition to classic modulators, epigenetic mechanisms regulate gene expression during development in response to environmental factors and in some diseases and have roles in steroid hormone action. Herein, we tested the hypothesis that DNA methylation plays a role in gene expression regulation in human endometrium in different hormonal milieux. High throughput, genome-wide DNA methylation profiling of endometrial samples in proliferative, early secretory, and midsecretory phases revealed dynamic DNA methylation patterns with segregation of proliferative from secretory phase samples by unsupervised cluster analysis of differentially methylated genes. Changes involved different frequencies of gain and loss of methylation within or outside CpG islands. Comparison of changes in transcriptomes and corresponding DNA methylomes from the same samples revealed association of DNA methylation and gene expression in a number of loci, some important in endometrial biology. Human endometrial stromal fibroblasts treated in vitro with estradiol and progesterone exhibited DNA methylation changes in several genes observed in proliferative and secretory phase tissues, respectively. Taken together, the data support the observation that epigenetic mechanisms are involved in gene expression regulation in human endometrium in different hormonal milieux, adding endometrium to a small number of normal adult tissues exhibiting dynamic DNA methylation. The data also raise the possibility that the interplay between steroid hormone and methylome dynamics regulates normal endometrial functions and, if abnormal, may result in endometrial dysfunction and associated disorders. PMID:24877562

Forensic Analysis of Human DNA from Samples Contamined with Bioweapons Agents

DTIC Science & Technology

2011-10-01

Forensic analysis of human DNA from samples contaminated with bioweapons agents Jason Timbers Kathryn Wright Royal Canadian Mounted...Police Forensic Science and Identification Service Prepared By: Royal Canadian Mounted Police RCMP Forensic Science Identification Services... Royal Canadian Mounted Police Forensic Science and Identification Service Prepared By: Royal Canadian Mounted Police RCMP Forensic Science

DNA Identification of Skeletal Remains from World War II Mass Graves Uncovered in Slovenia

PubMed Central

Marjanović, Damir; Durmić-Pašić, Adaleta; Bakal, Narcisa; Haverić, Sanin; Kalamujić, Belma; Kovačević, Lejla; Ramić, Jasmin; Pojskić, Naris; Škaro, Vedrana; Projić, Petar; Bajrović, Kasim; Hadžiselimović, Rifat; Drobnič, Katja; Huffine, Ed; Davoren, Jon; Primorac, Dragan

2007-01-01

Aim To present the joint effort of three institutions in the identification of human remains from the World War II found in two mass graves in the area of Škofja Loka, Slovenia. Methods The remains of 27 individuals were found in two small and closely located mass graves. The DNA was isolated from bone and teeth samples using either standard phenol/chloroform alcohol extraction or optimized Qiagen DNA extraction procedure. Some recovered samples required the employment of additional DNA purification methods, such as N-buthanol treatment. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex 16 kit was used to simultaneously amplify 15 short tandem repeat (STR) loci. Matching probabilities were estimated using the DNA View program. Results Out of all processed samples, 15 remains were fully profiled at all 15 STR loci. The other 12 profiles were partial. The least successful profile included 13 loci. Also, 69 referent samples (buccal swabs) from potential living relatives were collected and profiled. Comparison of victims' profile against referent samples database resulted in 4 strong matches. In addition, 5 other profiles were matched to certain referent samples with lower probability. Conclusion Our results show that more than 6 decades after the end of the World War II, DNA analysis may significantly contribute to the identification of the remains from that period. Additional analysis of Y-STRs and mitochondrial DNA (mtDNA) markers will be performed in the second phase of the identification project. PMID:17696306

DNA identification of human remains in Disaster Victim Identification (DVI): An efficient sampling method for muscle, bone, bone marrow and teeth.

PubMed

de Boer, Hans H; Maat, George J R; Kadarmo, D Aji; Widodo, Putut T; Kloosterman, Ate D; Kal, Arnoud J

2018-06-04

In disaster victim identification (DVI), DNA profiling is considered to be one of the most reliable and efficient means to identify bodies or separated body parts. This requires a post mortem DNA sample, and an ante mortem DNA sample of the presumed victim or their biological relative(s). Usually the collection of an adequate ante mortem sample is technically simple, but the acquisition of a good quality post mortem sample under unfavourable DVI circumstances is complicated due to the variable degree of preservation of the human remains and the high risk of DNA (cross) contamination. This paper provides the community with an efficient method to collect post-mortem DNA samples from muscle, bone, bone marrow and teeth, with a minimal risk of contamination. Our method has been applied in a recent, challenging DVI operation (i.e. the identification of the 298 victims of the MH17 airplane crash in 2014). 98,2% of the collected PM samples provided the DVI team with highly informative DNA genotyping results without the risk of contamination and consequent mistyping the victim's DNA. Moreover, the method is easy, cheap and quick. This paper provides the DVI community with a step-wise instructions with recommendations for the type of tissue to be sampled and the site of excision (preferably the upper leg). Although initially designed for DVI purposes, the method is also suited for the identification of individual victims. Copyright © 2018 Elsevier B.V. All rights reserved.

A DNA methylation map of human cancer at single base-pair resolution.

PubMed

Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

2017-10-05

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination.

Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols

PubMed Central

GARBIERI, Thais Francini; BROZOSKI, Daniel Thomas; DIONÍSIO, Thiago José; SANTOS, Carlos Ferreira; NEVES, Lucimara Teixeira das

2017-01-01

Abstract Saliva when compared to blood collection has the following advantages: it requires no specialized personnel for collection, allows for remote collection by the patient, is painless, well accepted by participants, has decreased risks of disease transmission, does not clot, can be frozen before DNA extraction and possibly has a longer storage time. Objective and Material and Methods This study aimed to compare the quantity and quality of human DNA extracted from saliva that was fresh or frozen for three, six and twelve months using five different DNA extraction protocols: protocol 1 – Oragene™ commercial kit, protocol 2 – QIAamp DNA mini kit, protocol 3 – DNA extraction using ammonium acetate, protocol 4 – Instagene™ Matrix and protocol 5 – Instagene™ Matrix diluted 1:1 using proteinase K and 1% SDS. Briefly, DNA was analyzed using spectrophotometry, electrophoresis and PCR. Results Results indicated that time spent in storage typically decreased the DNA quantity with the exception of protocol 1. The purity of DNA was generally not affected by storage times for the commercial based protocols, while the purity of the DNA samples extracted by the noncommercial protocols typically decreased when the saliva was stored longer. Only protocol 1 consistently extracted unfragmented DNA samples. In general, DNA samples extracted through protocols 1, 2, 3 and 4, regardless of storage time, were amplified by human specific primers whereas protocol 5 produced almost no samples that were able to be amplified by human specific primers. Depending on the protocol used, it was possible to extract DNA in high quantities and of good quality using whole saliva, and furthermore, for the purposes of DNA extraction, saliva can be reliably stored for relatively long time periods. Conclusions In summary, a complicated picture emerges when taking into account the extracted DNA’s quantity, purity and quality; depending on a given researchers needs, one protocol’s particular strengths and costs might be the deciding factor for its employment. PMID:28403355

Identification of Forensic Samples via Mitochondrial DNA in the Undergraduate Biochemistry Laboratory

NASA Astrophysics Data System (ADS)

Millard, Julie T.; Pilon, André M.

2003-04-01

A recent forensic approach for identification of unknown biological samples is mitochondrial DNA (mtDNA) sequencing. We describe a laboratory exercise suitable for an undergraduate biochemistry course in which the polymerase chain reaction is used to amplify a 440 base pair hypervariable region of human mtDNA from a variety of "crime scene" samples (e.g., teeth, hair, nails, cigarettes, envelope flaps, toothbrushes, and chewing gum). Amplification is verified via agarose gel electrophoresis and then samples are subjected to cycle sequencing. Sequence alignments are made via the program CLUSTAL W, allowing students to compare samples and solve the "crime."

Simultaneous analysis of six aldehyde-DNA adducts in salivary DNA of nonsmokers and smokers using stable isotope dilution liquid chromatography electrospray ionization-tandem mass spectrometry.

PubMed

Li, Xiangyu; Liu, Lujuan; Wang, Hongjuan; Chen, Jian; Zhu, Beibei; Chen, Huan; Hou, Hongwei; Hu, Qingyuan

2017-08-15

A stable method, using isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS), to simultaneously determine six aldehyde-DNA adducts was developed and applied to the analysis of human salivary DNA samples. The detection limit of these six DNA adducts was in the range of 0.006-0.014ng/mL and that of the quantification limit was 0.017-0.026ng/mL. The intra-day and inter-day precision of all aldehyde-DNA adducts was <10%. The analysis was completed within 25min. Additionally, a noninvasive technique was used to collect the DNA samples from human saliva. The new method was successfully applied for the analysis of salivary DNA of nonsmokers and smokers. Five aldehyde-DNA adducts were detected in both smoker and nonsmoker salivary DNA, while α-Acr-dG was not detected in all the samples. Among these detected DNA adducts, no significant differences were found between smoker and nonsmoker (p>0.05). This may due to the individual detoxifying differences or environmental and endogenous exposure. Our study provides a rapid and selective method to simultaneously detect six aldehyde-DNA adducts and to assess potential DNA damage induced by aldehydes. Copyright © 2017 Elsevier B.V. All rights reserved.

Presumptive Sources of Fecal Contamination in Four Tributaries to the New River Gorge National River, West Virginia, 2004

USGS Publications Warehouse

Mathes, Melvin V.; O'Brien, Tara L.; Strickler, Kriston M.; Hardy, Joshua J.; Schill, William B.; Lukasik, Jerzy; Scott, Troy M.; Bailey, David E.; Fenger, Terry L.

2007-01-01

Several methods were used to determine the sources of fecal contamination in water samples collected during September and October 2004 from four tributaries to the New River Gorge National River -- Arbuckle Creek, Dunloup Creek, Keeney Creek, and Wolf Creek. All four tributaries historically have had elevated levels of fecal coliform bacteria. The source-tracking methods used yielded various results, possibly because one or more methods failed. Sourcing methods used in this study included the detection of several human-specific and animal-specific biological or molecular markers, and library-dependent pulsed-field gel electrophoresis analysis that attempted to associate Escherichia coli bacteria obtained from water samples with animal sources by matching DNA-fragment banding patterns. Evaluation of the results of quality-control analysis indicated that pulsed-field gel electrophoresis analysis was unable to identify known-source bacteria isolates. Increasing the size of the known-source library did not improve the results for quality-control samples. A number of emerging methods, using markers in Enterococcus, human urine, Bacteroidetes, and host mitochondrial DNA, demonstrated some potential in associating fecal contamination with human or animal sources in a limited analysis of quality-control samples. All four of the human-specific markers were detected in water samples from Keeney Creek, a watershed with no centralized municipal wastewater-treatment facilities, thus indicating human sources of fecal contamination. The human-specific Bacteroidetes and host mitochondrial DNA markers were detected in water samples from Dunloup Creek, Wolf Creek, and to a lesser degree Arbuckle Creek. Results of analysis for wastewater compounds indicate that the September 27 sample from Arbuckle Creek contained numerous human tracer compounds likely from sewage. Dog, horse, chicken, and pig host mitochondrial DNA were detected in some of the water samples with the exception of the October 5 sample from Dunloup Creek. Cow, white-tailed deer, and Canada goose DNA were not detected in any of the samples collected from the four tributaries, despite the presence of these animals in the watersheds. Future studies with more rigorous quality-control analyses are needed to investigate the potential applicability and use of these emerging methods. Because many of the detections for the various methods could vary over time and with flow conditions, repeated sampling during both base flow and storm events would be necessary to more definitively determine the sources of fecal contamination for each watershed.

The Use and Effectiveness of Triple Multiplex System for Coding Region Single Nucleotide Polymorphism in Mitochondrial DNA Typing of Archaeologically Obtained Human Skeletons from Premodern Joseon Tombs of Korea

PubMed Central

Oh, Chang Seok; Lee, Soong Deok; Kim, Yi-Suk; Shin, Dong Hoon

2015-01-01

Previous study showed that East Asian mtDNA haplogroups, especially those of Koreans, could be successfully assigned by the coupled use of analyses on coding region SNP markers and control region mutation motifs. In this study, we tried to see if the same triple multiplex analysis for coding regions SNPs could be also applicable to ancient samples from East Asia as the complementation for sequence analysis of mtDNA control region. By the study on Joseon skeleton samples, we know that mtDNA haplogroup determined by coding region SNP markers successfully falls within the same haplogroup that sequence analysis on control region can assign. Considering that ancient samples in previous studies make no small number of errors in control region mtDNA sequencing, coding region SNP analysis can be used as good complimentary to the conventional haplogroup determination, especially of archaeological human bone samples buried underground over long periods. PMID:26345190

Performance evaluation of a mitogenome capture and Illumina sequencing protocol using non-probative, case-type skeletal samples: Implications for the use of a positive control in a next-generation sequencing procedure.

PubMed

Marshall, Charla; Sturk-Andreaggi, Kimberly; Daniels-Higginbotham, Jennifer; Oliver, Robert Sean; Barritt-Ross, Suzanne; McMahon, Timothy P

2017-11-01

Next-generation ancient DNA technologies have the potential to assist in the analysis of degraded DNA extracted from forensic specimens. Mitochondrial genome (mitogenome) sequencing, specifically, may be of benefit to samples that fail to yield forensically relevant genetic information using conventional PCR-based techniques. This report summarizes the Armed Forces Medical Examiner System's Armed Forces DNA Identification Laboratory's (AFMES-AFDIL) performance evaluation of a Next-Generation Sequencing protocol for degraded and chemically treated past accounting samples. The procedure involves hybridization capture for targeted enrichment of mitochondrial DNA, massively parallel sequencing using Illumina chemistry, and an automated bioinformatic pipeline for forensic mtDNA profile generation. A total of 22 non-probative samples and associated controls were processed in the present study, spanning a range of DNA quantity and quality. Data were generated from over 100 DNA libraries by ten DNA analysts over the course of five months. The results show that the mitogenome sequencing procedure is reliable and robust, sensitive to low template (one ng control DNA) as well as degraded DNA, and specific to the analysis of the human mitogenome. Haplotypes were overall concordant between NGS replicates and with previously generated Sanger control region data. Due to the inherent risk for contamination when working with low-template, degraded DNA, a contamination assessment was performed. The consumables were shown to be void of human DNA contaminants and suitable for forensic use. Reagent blanks and negative controls were analyzed to determine the background signal of the procedure. This background signal was then used to set analytical and reporting thresholds, which were designated at 4.0X (limit of detection) and 10.0X (limit of quantiation) average coverage across the mitogenome, respectively. Nearly all human samples exceeded the reporting threshold, although coverage was reduced in chemically treated samples resulting in a ∼58% passing rate for these poor-quality samples. A concordance assessment demonstrated the reliability of the NGS data when compared to known Sanger profiles. One case sample was shown to be mixed with a co-processed sample and two reagent blanks indicated the presence of DNA above the analytical threshold. This contamination was attributed to sequencing crosstalk from simultaneously sequenced high-quality samples to include the positive control. Overall this study demonstrated that hybridization capture and Illumina sequencing provide a viable method for mitogenome sequencing of degraded and chemically treated skeletal DNA samples, yet may require alternative measures of quality control. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Recovery of human DNA profiles from poached deer remains part 2: improved recovery protocol without the need for LCN analysis.

PubMed

Tobe, Shanan S; Bailey, Stuart; Govan, James; Welch, Lindsey A

2013-03-01

Although poaching is a common wildlife crime, the high and prohibitive cost of specialised animal testing means that many cases are left un-investigated. We previously described a novel approach to wildlife crime investigation that looked at the identification of human DNA on poached animal remains (Tobe, Govan and Welch, 2011). Human DNA was successfully isolated and amplified from simulated poaching incidents, however a low template protocol was required which made this method unsuitable for use in many laboratories. We now report on an optimised recovery and amplification protocol which removes the need for low template analysis. Samples from 10 deer (40 samples total - one from each leg) analysed in the original study were re-analysed in the current study with an additional 11 deer samples. Four samples analysed using Chelex did not show any results and a new method was devised whereby the available DNA was concentrated. By combining the DNA extracts from all tapings of the same deer remains followed by concentration, the recovered quantity of human DNA was found to be 29.5pg±43.2pg, 31× greater than the previous study. The use of the Investigator Decaplex SE (QIAGEN) STR kit provided better results in the form of more complete profiles than did the AmpFℓSTR® SGM Plus® kit at 30cycles (Applied Biosystems). Re-analysis of the samples from the initial study using the new, optimised protocol resulted in an average increase of 18% of recovered alleles. Over 17 samples, 71% of the samples analysed using the optimised protocol showed sufficient amplification for comparison to a reference profile and gave match probabilities ranging from 7.7690×10(-05) to 2.2706×10(-14). The removal of low template analysis means this optimised method provides evidence of high probative value and is suitable for immediate use in forensic laboratories. All methods and techniques used are standard and are compatible with current SOPs. As no high cost non-human DNA analysis is required the overall process is no more expensive than the investigation of other volume crime samples. The technique is suitable for immediate use in poaching incidents. Copyright © 2012 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.

Evidence for the presence of mutagenic arylamines in human breast milk and DNA adducts in exfoliated breast ductal epithelial cells.

PubMed

Thompson, Patricia A; DeMarini, David M; Kadlubar, Fred F; McClure, Gail Y; Brooks, Lance R; Green, Bridgett L; Fares, Manal Y; Stone, Angie; Josephy, P David; Ambrosone, Christine B

2002-01-01

Aromatic and heterocyclic amines are ubiquitous environmental mutagens present in combustion emissions, fried meats, and tobacco smoke, and are suspect human mammary carcinogens. To determine the presence of arylamines in breast tissue and fluid, we examined exfoliated breast ductal epithelial cells for DNA adducts and matched human milk samples for mutagenicity. Breast milk was obtained from 50 women who were 4-6 weeks postpartum, and exfoliated epithelial-cell DNA was evaluated for bulky, nonpolar DNA adducts by (32)P-postlabeling and thin-layer chromatography. Milk was processed by acid hydrolysis, and the extracted organics were examined in the standard plate-incorporation Ames Salmonella assay using primarily strain YG1024, which detects frameshift mutations and overexpresses aryl amine N-acetyltransferase. DNA adducts were identified in 66% of the specimens, and bulky adducts migrated in a pattern similar to that of 4-aminobiphenyl standards. The distribution of adducts did not vary by NAT2 genotype status. Of whole milk samples, 88% (22/25) had mutagenic activity. Among the samples for which we had both DNA adduct and mutagenicity data, 58% (14/19) of the samples with adducts were also mutagenic, and 85% (11/13) of the mutagenic samples had adducts. Quantitatively, no correlation was observed between the levels of adducts and the levels of mutagenicity. Separation of the milk showed that mutagenic activity was found in 69% of skimmed milk samples but in only 29% of the corresponding milk fat samples, suggesting that the breast milk mutagens were moderately polar molecules. Chemical fractionation showed that mutagenic activity was found in 67% (4/6) of the basic fractions but in only 33% (2/6) of acidic samples, indicating that the mutagens were primarily basic compounds, such as arylamines. Although pilot in nature, this study corroborates previous findings of significant levels of DNA adducts in breast tissue and mutagenicity in human breast milk and indicates that breast milk mutagens may be moderately polar basic compounds, such as arylamines.

Comparison of human papillomavirus DNA tests, liquid-based cytology and conventional cytology for the early detection of cervix uteri cancer.

PubMed

Girianelli, Vania R; Thuler, Luiz Claudio S; Szklo, Moyses; Donato, Alexandre; Zardo, Lucilia M G; Lozana, José A; Almeida Neto, Olimpio F; Carvalho, Aurenice C L; Matos, Jorge H; Figueiredo, Valeska

2006-12-01

To compare the performance of human papillomavirus DNA tests (samples collected by a healthcare professional and self-collected) and liquid-based cytology with conventional cytology in the detection of cervix uteri cancer and its precursor lesions. A cross-sectional study was carried out in 1777 women living in poor communities in Rio de Janeiro State, Brazil. Eligibility criteria included ages 25-59 years and not having had a Papanicolau test within at least 3 years prior to the study. Cytology (conventional or liquid-based) and human papillomavirus DNA (collected by a healthcare professional or self-collected) tests were performed using samples collected in a single visit. Women with abnormalities in at least one test and a systematic sample of 70 women with negative test results were referred to a colposcopic examination. Test readings were double-masked, and the outcome of interest was high-grade squamous intraepithelial lesion or worse. The pathology report was used as the gold standard. The prevalence of high-grade squamous intraepithelial lesion or worse was 2.0%. Human papillomavirus DNA test collected by a health professional alone or combined with conventional cytology had the highest sensitivity (91.4 and 97.1%, respectively). The highest specificity was found for conventional cytology (91.6%) and for a human papillomavirus DNA test collected by a healthcare professional (90.2%). On the basis of only test performance, the use of human papillomavirus DNA tests, alone or combined with cytology, would seem to be recommended. Its population-wide implementation, however, is conditional on a cost-effectiveness analysis.

No Evidence for Infection of UK Prostate Cancer Patients with XMRV, BK Virus, Trichomonas vaginalis or Human Papilloma Viruses

PubMed Central

Groom, Harriet C. T.; Warren, Anne Y.; Neal, David E.; Bishop, Kate N.

2012-01-01

The prevalence of specific infections in UK prostate cancer patients was investigated. Serum from 84 patients and 62 controls was tested for neutralisation of xenotropic murine leukaemia virus-related virus (XMRV) Envelope. No reactivity was found in the patient samples. In addition, a further 100 prostate DNA samples were tested for XMRV, BK virus, Trichomonas vaginalis and human papilloma viruses by nucleic acid detection techniques. Despite demonstrating DNA integrity and assay sensitivity, we failed to detect the presence of any of these agents in DNA samples, bar one sample that was weakly positive for HPV16. Therefore we conclude that these infections are absent in this typical cohort of men with prostate cancer. PMID:22470540

No evidence for infection of UK prostate cancer patients with XMRV, BK virus, Trichomonas vaginalis or human papilloma viruses.

PubMed

Groom, Harriet C T; Warren, Anne Y; Neal, David E; Bishop, Kate N

2012-01-01

The prevalence of specific infections in UK prostate cancer patients was investigated. Serum from 84 patients and 62 controls was tested for neutralisation of xenotropic murine leukaemia virus-related virus (XMRV) Envelope. No reactivity was found in the patient samples. In addition, a further 100 prostate DNA samples were tested for XMRV, BK virus, Trichomonas vaginalis and human papilloma viruses by nucleic acid detection techniques. Despite demonstrating DNA integrity and assay sensitivity, we failed to detect the presence of any of these agents in DNA samples, bar one sample that was weakly positive for HPV16. Therefore we conclude that these infections are absent in this typical cohort of men with prostate cancer.

Developmental validation of the HIrisPlex system: DNA-based eye and hair colour prediction for forensic and anthropological usage.

PubMed

Walsh, Susan; Chaitanya, Lakshmi; Clarisse, Lindy; Wirken, Laura; Draus-Barini, Jolanta; Kovatsi, Leda; Maeda, Hitoshi; Ishikawa, Takaki; Sijen, Titia; de Knijff, Peter; Branicki, Wojciech; Liu, Fan; Kayser, Manfred

2014-03-01

Forensic DNA Phenotyping or 'DNA intelligence' tools are expected to aid police investigations and find unknown individuals by providing information on externally visible characteristics of unknown suspects, perpetrators and missing persons from biological samples. This is especially useful in cases where conventional DNA profiling or other means remain non-informative. Recently, we introduced the HIrisPlex system, capable of predicting both eye and hair colour from DNA. In the present developmental validation study, we demonstrate that the HIrisPlex assay performs in full agreement with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines providing an essential prerequisite for future HIrisPlex applications to forensic casework. The HIrisPlex assay produces complete profiles down to only 63 pg of DNA. Species testing revealed human specificity for a complete HIrisPlex profile, while only non-human primates showed the closest full profile at 20 out of the 24 DNA markers, in all animals tested. Rigorous testing of simulated forensic casework samples such as blood, semen, saliva stains, hairs with roots as well as extremely low quantity touch (trace) DNA samples, produced complete profiles in 88% of cases. Concordance testing performed between five independent forensic laboratories displayed consistent reproducible results on varying types of DNA samples. Due to its design, the assay caters for degraded samples, underlined here by results from artificially degraded DNA and from simulated casework samples of degraded DNA. This aspect was also demonstrated previously on DNA samples from human remains up to several hundreds of years old. With this paper, we also introduce enhanced eye and hair colour prediction models based on enlarged underlying databases of HIrisPlex genotypes and eye/hair colour phenotypes (eye colour: N = 9188 and hair colour: N = 1601). Furthermore, we present an online web-based system for individual eye and hair colour prediction from full and partial HIrisPlex DNA profiles. By demonstrating that the HIrisPlex assay is fully compatible with the SWGDAM guidelines, we provide the first forensically validated DNA test system for parallel eye and hair colour prediction now available to forensic laboratories for immediate casework application, including missing person cases. Given the robustness and sensitivity described here and in previous work, the HIrisPlex system is also suitable for analysing old and ancient DNA in anthropological and evolutionary studies. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Recovery of human DNA profiles from poached deer remains: a feasibility study.

PubMed

Tobe, Shanan S; Govan, James; Welch, Lindsey A

2011-12-01

Poaching is a crime that occurs worldwide and can be extremely difficult to investigate and prosecute due to the nature of the evidence available. If a species is protected by international legislation such as the Convention on International Trade in Endangered Species of Wild Fauna and Flora then simply possessing any part of that species is illegal. Previous studies have focused on the identification of endangered species in cases of potential poaching. Difficulties arise if the poached animal is not endangered. Species such as deer have hunting seasons whereby they can legally be hunted however poaching is the illegal take of deer, irrespective of season. Therefore, identification of deer alone has little probative value as samples could have originated from legal hunting activities in season. After a deer is hunted it is usual to remove the innards, head and lower limbs. The limbs are removed through manual force and represent a potential source of human touch DNA. We investigate the potential to recover and profile human autosomal DNA from poached deer remains. Samples from the legs of ten culled deer were obtained (40 in total) using minitapes. DNA from samples was extracted, quantified and amplified to determine if it would be possible to recover human STR profiles. Low quantification data led to the use of an extended PCR cycling protocol of 34 cycles. Samples from seven deer amplified, however some samples were excluded from further analysis due to 'drop in' alleles or the low level of successfully amplified loci. Samples from five deer could be further analysed and gave match probabilities ranging from 6.37×10(-3) to 9.53×10(-11). This study demonstrates the potential of recovering human touch DNA from poached animal remains. There is the potential for this test to be used in relation to other species of poached remains or other types of wildlife crimes. This is the first time, to our knowledge, that human STR profiling has been successfully applied to touch DNA in regards to simulated wildlife crime. Copyright © 2011 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.

Specificity of a Bacteroides thetaiotaomicron marker for human feces

USGS Publications Warehouse

Carson, C.A.; Christiansen, J.M.; Yampara-Iquise, H.; Benson, V.W.; Baffaut, C.; Davis, J.V.; Broz, R.R.; Kurtz, W.B.; Rogers, W.M.; Fales, W.H.

2005-01-01

A bacterial primer set, known to produce a 542-bp amplicon specific for Bacteroides thetaiotaomicron, generated this product in PCR with 1 ng of extracted DNA from 92% of 25 human fecal samples, 100% of 20 sewage samples, and 16% of 31 dog fecal samples. The marker was not detected in 1 ng of fecal DNA from 61 cows, 35 horses, 44 pigs, 24 chickens, 29 turkeys, and 17 geese. Copyright ?? 2005, American Society for Microbiology. All Rights Reserved.

Use of Sequenom Sample ID Plus® SNP Genotyping in Identification of FFPE Tumor Samples

PubMed Central

Miller, Jessica K.; Buchner, Nicholas; Timms, Lee; Tam, Shirley; Luo, Xuemei; Brown, Andrew M. K.; Pasternack, Danielle; Bristow, Robert G.; Fraser, Michael; Boutros, Paul C.; McPherson, John D.

2014-01-01

Short tandem repeat (STR) analysis, such as the AmpFlSTR® Identifiler® Plus kit, is a standard, PCR-based human genotyping method used in the field of forensics. Misidentification of cell line and tissue DNA can be costly if not detected early; therefore it is necessary to have quality control measures such as STR profiling in place. A major issue in large-scale research studies involving archival formalin-fixed paraffin embedded (FFPE) tissues is that varying levels of DNA degradation can result in failure to correctly identify samples using STR genotyping. PCR amplification of STRs of several hundred base pairs is not always possible when DNA is degraded. The Sample ID Plus® panel from Sequenom allows for human DNA identification and authentication using SNP genotyping. In comparison to lengthy STR amplicons, this multiplexing PCR assay requires amplification of only 76–139 base pairs, and utilizes 47 SNPs to discriminate between individual samples. In this study, we evaluated both STR and SNP genotyping methods of sample identification, with a focus on paired FFPE tumor/normal DNA samples intended for next-generation sequencing (NGS). The ability to successfully validate the identity of FFPE samples can enable cost savings by reducing rework. PMID:24551080

Use of Sequenom sample ID Plus® SNP genotyping in identification of FFPE tumor samples.

PubMed

Miller, Jessica K; Buchner, Nicholas; Timms, Lee; Tam, Shirley; Luo, Xuemei; Brown, Andrew M K; Pasternack, Danielle; Bristow, Robert G; Fraser, Michael; Boutros, Paul C; McPherson, John D

2014-01-01

Short tandem repeat (STR) analysis, such as the AmpFlSTR® Identifiler® Plus kit, is a standard, PCR-based human genotyping method used in the field of forensics. Misidentification of cell line and tissue DNA can be costly if not detected early; therefore it is necessary to have quality control measures such as STR profiling in place. A major issue in large-scale research studies involving archival formalin-fixed paraffin embedded (FFPE) tissues is that varying levels of DNA degradation can result in failure to correctly identify samples using STR genotyping. PCR amplification of STRs of several hundred base pairs is not always possible when DNA is degraded. The Sample ID Plus® panel from Sequenom allows for human DNA identification and authentication using SNP genotyping. In comparison to lengthy STR amplicons, this multiplexing PCR assay requires amplification of only 76-139 base pairs, and utilizes 47 SNPs to discriminate between individual samples. In this study, we evaluated both STR and SNP genotyping methods of sample identification, with a focus on paired FFPE tumor/normal DNA samples intended for next-generation sequencing (NGS). The ability to successfully validate the identity of FFPE samples can enable cost savings by reducing rework.

Validation of DESS as a DNA Preservation Method for the Detection of Strongyloides spp. in Canine Feces.

PubMed

Beknazarova, Meruyert; Millsteed, Shelby; Robertson, Gemma; Whiley, Harriet; Ross, Kirstin

2017-06-09

Strongyloides stercoralis is a gastrointestinal parasitic nematode with a life cycle that includes free-living and parasitic forms. For both clinical (diagnostic) and environmental evaluation, it is important that we can detect Strongyloides spp. in both human and non-human fecal samples. Real-time PCR is the most feasible method for detecting the parasite in both clinical and environmental samples that have been preserved. However, one of the biggest challenges with PCR detection is DNA degradation during the postage time from rural and remote areas to the laboratory. This study included a laboratory assessment and field validation of DESS (dimethyl sulfoxide, disodium EDTA, and saturated NaCl) preservation of Strongyloides spp. DNA in fecal samples. The laboratory study investigated the capacity of 1:1 and 1:3 sample to DESS ratios to preserve Strongyloides ratti in spike canine feces. It was found that both ratios of DESS significantly prevented DNA degradation compared to the untreated sample. This method was then validated by applying it to the field-collected canine feces and detecting Strongyloides DNA using PCR. A total of 37 canine feces samples were collected and preserved in the 1:3 ratio (sample: DESS) and of these, 17 were positive for Strongyloides spp. The study shows that both 1:1 and 1:3 sample to DESS ratios were able to preserve the Strongyloides spp. DNA in canine feces samples stored at room temperature for up to 56 days. This DESS preservation method presents the most applicable and feasible method for the Strongyloides DNA preservation in field-collected feces.

A DNA methylation map of human cancer at single base-pair resolution

PubMed Central

Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

2017-01-01

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination. PMID:28581523

LAMPhimerus: A novel LAMP assay for detecting Amphimerus sp. DNA in human stool samples

PubMed Central

Calvopiña, Manuel; Fontecha-Cuenca, Cristina; Sugiyama, Hiromu; Sato, Megumi; López Abán, Julio; Vicente, Belén; Muro, Antonio

2017-01-01

Background Amphimeriasis is a fish-borne disease caused by the liver fluke Amphimerus spp. that has recently been reported as endemic in the tropical Pacific side of Ecuador with a high prevalence in humans and domestic animals. The diagnosis is based on the stool examination to identify parasite eggs, but it lacks sensitivity. Additionally, the morphology of the eggs may be confounded with other liver and intestinal flukes. No immunological or molecular methods have been developed to date. New diagnostic techniques for specific and sensitive detection of Amphimerus spp. DNA in clinical samples are needed. Methodology/Principal findings A LAMP targeting a sequence of the Amphimerus sp. internal transcribed spacer 2 region was designed. Amphimerus sp. DNA was obtained from adult worms recovered from animals and used to optimize the molecular assays. Conventional PCR was performed using outer primers F3-B3 to verify the proper amplification of the Amphimerus sp. DNA target sequence. LAMP was optimized using different reaction mixtures and temperatures, and it was finally set up as LAMPhimerus. The specificity and sensitivity of both PCR and LAMP were evaluated. The detection limit was 1 pg of genomic DNA. Field testing was done using 44 human stool samples collected from localities where fluke is endemic. Twenty-five samples were microscopy positive for Amphimerus sp. eggs detection. In molecular testing, PCR F3-B3 was ineffective when DNA from fecal samples was used. When testing all human stool samples included in our study, the diagnostic parameters for the sensitivity and specificity were calculated for our LAMPhimerus assay, which were 76.67% and 80.77%, respectively. Conclusions/Significance We have developed and evaluated, for the first time, a specific and sensitive LAMP assay for detecting Amphimerus sp. in human stool samples. The procedure has been named LAMPhimerus method and has the potential to be adapted for field diagnosis and disease surveillance in amphimeriasis-endemic areas. Future large-scale studies will assess the applicability of this novel LAMP assay. PMID:28628614

Diagnosis of amphimeriasis by LAMPhimerus assay in human stool samples long-term storage onto filter paper

PubMed Central

Calvopiña, Manuel; Buendía-Sánchez, María; López-Abán, Julio; Vicente, Belén; Muro, Antonio

2018-01-01

Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., has recently been reported as an emerging disease affecting an indigenous Ameridian group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was the microscopic detection of eggs from the parasite in patients' stool samples with very low sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG in human sera and a molecular method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be much more sensitive than classical parasitological methods for amphimeriasis diagnosis using human stool samples for analysis. The objective of this work is to demonstrate the feasibility of using dried stool samples on filter paper as source of DNA in combination with the effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples collected from Chachi population were spread as thin layer onto common filter paper for easily transportation to our laboratory and stored at room temperature for one year until DNA extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity werecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We demonstrate, for the first time, that common filter paper is useful for easy collection and long-term storage of human stool samples for later DNA extraction and molecular analysis of human-parasitic trematode eggs. This simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to beused as an effective molecular large-scale screening test for amphimeriasis-endemic areas. PMID:29444135

Rapid detection of Opisthorchis viverrini and Strongyloides stercoralis in human fecal samples using a duplex real-time PCR and melting curve analysis.

PubMed

Janwan, Penchom; Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Anamnart, Witthaya; Maleewong, Wanchai

2011-12-01

Human opisthorchiasis caused by the liver fluke Opisthorchis viverrini is an endemic disease in Southeast Asian countries including the Lao People's Democratic Republic, Cambodia, Vietnam, and Thailand. Infection with the soil-transmitted roundworm Strongyloides stercoralis is an important problem worldwide. In some areas, both parasitic infections are reported as co-infections. A duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis was developed for the rapid detection of O. viverrini and S. stercoralis in human fecal samples. Duplex real-time FRET PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two genera of DNA elements: the 162 bp pOV-A6 DNA sequence specific to O. viverrini and the 244 bp 18S rRNA sequence specific to S. stercoralis, and two pairs of specific fluorophore-labeled probes. Both O. viverrini and S. stercoralis can be differentially detected in infected human fecal samples by this process through their different fluorescence channels and melting temperatures. Detection limit of the method was as little as two O. viverrini eggs and four S. stercoralis larvae in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasite materials, as well as from the DNA of human leukocytes and other control parasites. The technique showed 100% sensitivity and specificity. The introduced duplex real-time FRET PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The method is important for simultaneous detection especially in areas where both parasites overlap incidence and is useful as the screening tool in the returning travelers and immigrants to industrialized countries where number of samples in the diagnostic units will become increasing.

DNA quality and quantity from up to 16 years old post-mortem blood stored on FTA cards.

PubMed

Rahikainen, Anna-Liina; Palo, Jukka U; de Leeuw, Wiljo; Budowle, Bruce; Sajantila, Antti

2016-04-01

Blood samples preserved on FTA cards offer unique opportunities for genetic research. DNA recovered from these cards should be stable for long periods of time. However, it is not well established as how well the DNA stored on FTA card for substantial time periods meets the demands of forensic or genomic DNA analyses and especially so for from post-mortem (PM) samples in which the quality can vary upon initial collection. The aim of this study was to evaluate the time-dependent degradation on DNA quality and quantity extracted from up to 16 years old post-mortem bloodstained FTA cards. Four random FTA samples from eight time points spanning 1998 to 2013 (n=32) were collected and extracted in triplicate. The quantity and quality of the extracted DNA samples were determined with Quantifiler(®) Human Plus (HP) Quantification kit. Internal sample and sample-to-sample variation were evaluated by comparing recovered DNA yields. The DNA from the triplicate samplings were subsequently combined and normalized for further analysis. The practical effect of degradation on DNA quality was evaluated from normalized samples both with forensic and pharmacogenetic target markers. Our results suggest that (1) a PM change, e.g. blood clotting prior to sampling, affects the recovered DNA yield, creating both internal and sample-to-sample variation; (2) a negative correlation between the FTA card storage time and DNA quantity (r=-0.836 at the 0.01 level) was observed; (3) a positive correlation (r=0.738 at the level 0.01) was found between FTA card storage time and degradation levels. However, no inhibition was observed with the method used. The effect of degradation was manifested clearly with functional applications. Although complete STR-profiles were obtained for all samples, there was evidence of degradation manifested as decreased peak heights in the larger-sized amplicons. Lower amplification success was notable with the large 5.1 kb CYP2D6 gene fragment which strongly supports degradation of the stored samples. According to our results, DNA stored on FTA cards is rather stable over a long time period. DNA extracted from this storage medium can be used as human identification purposes as the method used is sufficiently sensitive and amplicon sizes tend to be <400 bp. However, DNA integrity was affected during storage. This effect should be taken into account depending on the intended application especially if high quality DNA and long PCR amplicons are required. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Fragmentation of contaminant and endogenous DNA in ancient samples determined by shotgun sequencing; prospects for human palaeogenomics.

PubMed

García-Garcerà, Marc; Gigli, Elena; Sanchez-Quinto, Federico; Ramirez, Oscar; Calafell, Francesc; Civit, Sergi; Lalueza-Fox, Carles

2011-01-01

Despite the successful retrieval of genomes from past remains, the prospects for human palaeogenomics remain unclear because of the difficulty of distinguishing contaminant from endogenous DNA sequences. Previous sequence data generated on high-throughput sequencing platforms indicate that fragmentation of ancient DNA sequences is a characteristic trait primarily arising due to depurination processes that create abasic sites leading to DNA breaks. METHODOLOGY/PRINCIPALS FINDINGS: To investigate whether this pattern is present in ancient remains from a temperate environment, we have 454-FLX pyrosequenced different samples dated between 5,500 and 49,000 years ago: a bone from an extinct goat (Myotragus balearicus) that was treated with a depurinating agent (bleach), an Iberian lynx bone not subjected to any treatment, a human Neolithic sample from Barcelona (Spain), and a Neandertal sample from the El Sidrón site (Asturias, Spain). The efficiency of retrieval of endogenous sequences is below 1% in all cases. We have used the non-human samples to identify human sequences (0.35 and 1.4%, respectively), that we positively know are contaminants. We observed that bleach treatment appears to create a depurination-associated fragmentation pattern in resulting contaminant sequences that is indistinguishable from previously described endogenous sequences. Furthermore, the nucleotide composition pattern observed in 5' and 3' ends of contaminant sequences is much more complex than the flat pattern previously described in some Neandertal contaminants. Although much research on samples with known contaminant histories is needed, our results suggest that endogenous and contaminant sequences cannot be distinguished by the fragmentation pattern alone.

Extreme-Depth Re-sequencing of Mitochondrial DNA Finds No Evidence of Paternal Transmission in Humans.

PubMed

Pyle, Angela; Hudson, Gavin; Wilson, Ian J; Coxhead, Jonathan; Smertenko, Tania; Herbert, Mary; Santibanez-Koref, Mauro; Chinnery, Patrick F

2015-05-01

Recent reports have questioned the accepted dogma that mammalian mitochondrial DNA (mtDNA) is strictly maternally inherited. In humans, the argument hinges on detecting a signature of inter-molecular recombination in mtDNA sequences sampled at the population level, inferring a paternal source for the mixed haplotypes. However, interpreting these data is fraught with difficulty, and direct experimental evidence is lacking. Using extreme-high depth mtDNA re-sequencing up to ~1.2 million-fold coverage, we find no evidence that paternal mtDNA haplotypes are transmitted to offspring in humans, thus excluding a simple dilution mechanism for uniparental transmission of mtDNA present in all healthy individuals. Our findings indicate that an active mechanism eliminates paternal mtDNA which likely acts at the molecular level.

Extreme-Depth Re-sequencing of Mitochondrial DNA Finds No Evidence of Paternal Transmission in Humans

PubMed Central

Pyle, Angela; Hudson, Gavin; Wilson, Ian J.; Coxhead, Jonathan; Smertenko, Tania; Herbert, Mary; Santibanez-Koref, Mauro; Chinnery, Patrick F.

2015-01-01

Recent reports have questioned the accepted dogma that mammalian mitochondrial DNA (mtDNA) is strictly maternally inherited. In humans, the argument hinges on detecting a signature of inter-molecular recombination in mtDNA sequences sampled at the population level, inferring a paternal source for the mixed haplotypes. However, interpreting these data is fraught with difficulty, and direct experimental evidence is lacking. Using extreme-high depth mtDNA re-sequencing up to ~1.2 million-fold coverage, we find no evidence that paternal mtDNA haplotypes are transmitted to offspring in humans, thus excluding a simple dilution mechanism for uniparental transmission of mtDNA present in all healthy individuals. Our findings indicate that an active mechanism eliminates paternal mtDNA which likely acts at the molecular level. PMID:25973765

An Alu-based, MGB Eclipse real-time PCR method for quantitation of human DNA in forensic samples.

PubMed

Nicklas, Janice A; Buel, Eric

2005-09-01

The forensic community needs quick, reliable methods to quantitate human DNA in crime scene samples to replace the laborious and imprecise slot blot method. A real-time PCR based method has the possibility of allowing development of a faster and more quantitative assay. Alu sequences are primate-specific and are found in many copies in the human genome, making these sequences an excellent target or marker for human DNA. This paper describes the development of a real-time Alu sequence-based assay using MGB Eclipse primers and probes. The advantages of this assay are simplicity, speed, less hands-on-time and automated quantitation, as well as a large dynamic range (128 ng/microL to 0.5 pg/microL).

Self-sampling for human papillomavirus DNA detection: a preliminary study of compliance and feasibility in BOLIVIA.

PubMed

Surriabre, Pedro; Allende, Gustavo; Prado, Marcela; Cáceres, Leyddy; Bellot, Diego; Torrico, Andrea; Ustariz, Karina; Rojas, Shirley; Barriga, Jaime; Calle, Pamela; Villarroel, Ligia; Yañez, Rosse Mary; Baay, Marc; Rodriguez, Patricia; Fontaine, Véronique

2017-12-22

Cervical cancer incidence and mortality rates in Bolivia are among the highest in Latin America. This investigation aims to evaluate the possibility of using simple devices, e.g. a cotton swab and a glass slide, for self-sampling in order to detect human papillomavirus (HPV) DNA by PCR in cervico-vaginal cells. In the first phase of our study we evaluated the use of a glass slide as a transport medium for cervical cells. A physician took paired-cervical samples from 235 women. One sample was transported in Easyfix® solution and the other sample was smeared over a glass slide. Both were further analyzed and compared for human DNA recovery and HPV detection. A kappa value was determined to evaluate the agreement between the HPV DNA detection rates. In the second phase of the study, 222 women from the urban, peri-urban and rural regions of Cochabamba were requested to perform self-sampling using the following devices: a cotton swab combined with a glass slide, and a vaginal tampon. Women gave their opinion about the self-sampling technique. Finally, the agreement for high risk-HPV detection between self- and physician-collected samples was performed in 201 samples in order to evaluate the self-sampling technique. Firstly, the comparison between Easyfix® solution and the glass slide to transport clinical samples gave a good agreement for HPV DNA detection (κ = 0.71, 95% CI 0.60-0.81). Secondly, self-sampling, especially with cotton swab combined with glass slide, would generally be preferred over clinician sampling for a screening program based on HPV detection. Finally, we showed a good agreement between self- and physician collected samples for high risk-HPV detection (κ = 0.71, 95% CI 0.55-0.88). Simple devices such as a cotton swab and a glass slide can be used to perform self-sampling and HPV DNA detection. Furthermore, most Bolivian women preferred self-sampling over clinician-sampling for cervical cancer screening.

DNA typing of ancient parasite eggs from environmental samples identifies human and animal worm infections in Viking-age settlement.

PubMed

Søe, Martin Jensen; Nejsum, Peter; Fredensborg, Brian Lund; Kapel, Christian Moliin Outzen

2015-02-01

Ancient parasite eggs were recovered from environmental samples collected at a Viking-age settlement in Viborg, Denmark, dated 1018-1030 A.D. Morphological examination identified Ascaris sp., Trichuris sp., and Fasciola sp. eggs, but size and shape did not allow species identification. By carefully selecting genetic markers, PCR amplification and sequencing of ancient DNA (aDNA) isolates resulted in identification of: the human whipworm, Trichuris trichiura , using SSUrRNA sequence homology; Ascaris sp. with 100% homology to cox1 haplotype 07; and Fasciola hepatica using ITS1 sequence homology. The identification of T. trichiura eggs indicates that human fecal material is present and, hence, that the Ascaris sp. haplotype 07 was most likely a human variant in Viking-age Denmark. The location of the F. hepatica finding suggests that sheep or cattle are the most likely hosts. Further, we sequenced the Ascaris sp. 18S rRNA gene in recent isolates from humans and pigs of global distribution and show that this is not a suited marker for species-specific identification. Finally, we discuss ancient parasitism in Denmark and the implementation of aDNA analysis methods in paleoparasitological studies. We argue that when employing species-specific identification, soil samples offer excellent opportunities for studies of human parasite infections and of human and animal interactions of the past.

Development of a Multiplex Single Base Extension Assay for Mitochondrial DNA Haplogroup Typing

PubMed Central

Nelson, Tahnee M.; Just, Rebecca S.; Loreille, Odile; Schanfield, Moses S.; Podini, Daniele

2007-01-01

Aim To provide a screening tool to reduce time and sample consumption when attempting mtDNA haplogroup typing. Methods A single base primer extension assay was developed to enable typing, in a single reaction, of twelve mtDNA haplogroup specific polymorphisms. For validation purposes a total of 147 samples were tested including 73 samples successfully haplogroup typed using mtDNA control region (CR) sequence data, 21 samples inconclusively haplogroup typed by CR data, 20 samples previously haplogroup typed using restriction fragment length polymorphism (RFLP) analysis, and 31 samples of known ancestral origin without previous haplogroup typing. Additionally, two highly degraded human bones embalmed and buried in the early 1950s were analyzed using the single nucleotide polymorphisms (SNP) multiplex. Results When the SNP multiplex was used to type the 96 previously CR sequenced specimens, an increase in haplogroup or macrohaplogroup assignment relative to conventional CR sequence analysis was observed. The single base extension assay was also successfully used to assign a haplogroup to decades-old, embalmed skeletal remains dating to World War II. Conclusion The SNP multiplex was successfully used to obtain haplogroup status of highly degraded human bones, and demonstrated the ability to eliminate possible contributors. The SNP multiplex provides a low-cost, high throughput method for typing of mtDNA haplogroups A, B, C, D, E, F, G, H, L1/L2, L3, M, and N that could be useful for screening purposes for human identification efforts and anthropological studies. PMID:17696300

Sequence of the cDNA of a human dihydrodiol dehydrogenase isoform (AKR1C2) and tissue distribution of its mRNA.

PubMed Central

Shiraishi, H; Ishikura, S; Matsuura, K; Deyashiki, Y; Ninomiya, M; Sakai, S; Hara, A

1998-01-01

Human liver contains three isoforms (DD1, DD2 and DD4) of dihydrodiol dehydrogenase with 20alpha- or 3alpha-hydroxysteroid dehydrogenase activity; the dehydrogenases belong to the aldo-oxo reductase (AKR) superfamily. cDNA species encoding DD1 and DD4 have been identified. However, four cDNA species with more than 99% sequence identity have been cloned and are compatible with a partial amino acid sequence of DD2. In this study we have isolated a cDNA clone encoding DD2, which was confirmed by comparison of the properties of the recombinant and hepatic enzymes. This cDNA showed differences of one, two, four and five nucleotides from the previously reported four cDNA species for a dehydrogenase of human colon carcinoma HT29 cells, human prostatic 3alpha-hydroxysteroid dehydrogenase, a human liver 3alpha-hydroxysteroid dehydrogenase-like protein and chlordecone reductase-like protein respectively. Expression of mRNA species for the five similar cDNA species in 20 liver samples and 10 other different tissue samples was examined by reverse transcriptase-mediated PCR with specific primers followed by diagnostic restriction with endonucleases. All the tissues expressed only one mRNA species corresponding to the newly identified cDNA for DD2: mRNA transcripts corresponding to the other cDNA species were not detected. We suggest that the new cDNA is derived from the principal gene for DD2, which has been named AKR1C2 by a new nomenclature for the AKR superfamily. It is possible that some of the other cDNA species previously reported are rare allelic variants of this gene. PMID:9716498

Preliminary perspectives on DNA collection in anti-human trafficking efforts.

PubMed

Katsanis, Sara H; Kim, Joyce; Minear, Mollie A; Chandrasekharan, Subhashini; Wagner, Jennifer K

2014-01-01

Forensic DNA methodologies have potential applications in the investigation of human trafficking cases. DNA and relationship testing may be useful for confirmation of biological relationship claims in immigration, identification of trafficked individuals who are missing persons, and family reunification of displaced individuals after mass disasters and conflicts. As these applications rely on the collection of DNA from non-criminals and potentially vulnerable individuals, questions arise as to how to address the ethical challenges of collection, security, and privacy of collected samples and DNA profiles. We administered a survey targeted to victims' advocates to gain preliminary understanding of perspectives regarding human trafficking definitions, DNA and sex workers, and perceived trust of authorities potentially involved in DNA collection. We asked respondents to consider the use of DNA for investigating adoption fraud, sex trafficking, and post-conflict child soldier cases. We found some key differences in perspectives on defining what qualifies as "trafficking." When we varied terminology between "sex worker" and "sex trafficking victim" we detected differences in perception on which authorities can be trusted. Respondents were supportive of the hypothetical models proposed to collect DNA. Most were favorable of DNA specimens being controlled by an authority outside of law enforcement. Participants voiced concerns focused on privacy, misuse of DNA samples and data, unintentional harms, data security, and infrastructure. These preliminary data indicate that while there is perceived value in programs to use DNA for investigating cases of human trafficking, these programs may need to consider levels of trust in authorities as their logistics are developed and implemented.

Genetic homogeneity among Leishmania (Leishmania) infantum isolates from dog and human samples in Belo Horizonte Metropolitan Area (BHMA), Minas Gerais, Brazil.

PubMed

da Silva, Thais Almeida Marques; Gomes, Luciana Inácia; Oliveira, Edward; Coura-Vital, Wendel; Silva, Letícia de Azevedo; Pais, Fabiano Sviatopolk-Mirsky; Ker, Henrique Gama; Reis, Alexandre Barbosa; Rabello, Ana; Carneiro, Mariangela

2015-04-15

Certain municipalities in the Belo Horizonte Metropolitan Area (BHMA), Minas Gerais, Brazil, have the highest human visceral leishmaniasis (VL) mortality rates in the country and also demonstrate high canine seropositivity. In Brazil, the etiologic agent of VL is Leishmania (Leishmania) infantum. The aim of this study was to evaluate the intraspecific genetic variability of parasites from humans and from dogs with different clinical forms of VL in five municipalities of BHMA using PCR-RFLP and two target genes: kinetoplast DNA (kDNA) and gp63. In total, 45 samples of DNA extracted from clinical samples (n = 35) or L. infantum culture (n = 10) were evaluated. These samples originated from three groups: adults (with or without Leishmania/HIV co-infection; n = 14), children (n = 18) and dogs (n = 13). The samples were amplified for the kDNA target using the MC1 and MC2 primers (447 bp), while the Sg1 and Sg2 (1330 bp) primers were used for the gp63 glycoprotein target gene. The restriction enzyme patterns of all the samples tested were monomorphic. These findings reveal a high degree of genetic homogeneity for the evaluated gene targets among L. infantum samples isolated from different hosts and representing different clinical forms of VL in the municipalities of BHMA studied.

DNA Identification of Commingled Human Remains from the Cemetery Relocated by Flooding in Central Bosnia and Herzegovina.

PubMed

Čakar, Jasmina; Pilav, Amela; Džehverović, Mirela; Ahatović, Anesa; Haverić, Sanin; Ramić, Jasmin; Marjanović, Damir

2018-01-01

The floods in Bosnia and Herzegovina in May 2014 caused landslides all over the country. In the small village of Šerići, near the town of Zenica, a landslide destroyed the local cemetery, relocated graves, and commingled skeletal remains. As the use of other physical methods of identification (facial recognition, fingerprint analysis, dental analysis, etc.) was not possible, DNA analysis was applied. DNA was isolated from 20 skeletal remains (bone and tooth samples) and six reference samples (blood from living relatives) and amplified using PowerPlex ® Fusion and PowerPlex ® Y23 kits. DNA profiles were generated for all reference samples and 17 skeletal remains. A statistical analysis (calculation of paternity, maternity, and sibling indexes and matching probabilities) resulted in 10 positive identifications. In this study, 5 individuals were identified based on one reference sample. This has once again demonstrated the significance of DNA analysis in resolving the most complicated cases, such as the identification of commingled human skeletal remains. © 2017 American Academy of Forensic Sciences.

Occurrence of viral DNA in paired samples of corneal rim and cornea preservation fluid.

PubMed

Broniek, G; Langwińska-Wośko, E; Sybilska, M; Szaflik, J P; Przybylski, M; Wróblewska, M

2017-04-01

Corneal transplants have one of the highest success rates among all transplantological procedures. Corneas intended for transplantation are stored in a preservation fluid, which is then tested for bacterial and fungal infections. Among all analyses of infectious complications following corneal transplants, infections caused by bacteria or fungi are the most prominent. Surprisingly, however, apart from a few publications, there is a lack of data regarding the occurrence of viruses in donor corneas and the risk of transmitting these to their recipients. The intention of this research was therefore to determine the frequency with which human herpesvirus 1 (HHV-1), human herpesvirus 2 (HHV-2), and human adenovirus (HAdV) occur in transplanted corneal tissue, as well as in samples of preservation fluid. The study comprised 57 paired samples, with each pair consisting of a fragment of the corneal tissue remaining after its trepanation for transplantation surgery and a sample of corneal preservation fluid. Sample pairs were all tested for the presence of the DNA of three viruses (HHV-1, HHV-2, and HAdV) using real time PCR technique. Viral DNA was found in three of the tested corneas-HHV-1 DNA in one paired sample (1.8%) and adenovirus DNA in two single samples (3.5%). We postulate that virological testing of corneas for transplantation should be considered, particularly in the case of donors with increased risk factors for herpesvirus and adenovirus reactivation. J. Med. Virol. 89:732-736, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

[Macrophages in human semen].

PubMed

Bouvet, Beatriz Reina; Brufman, Adriana Silvia; Paparella, Cecilia Vicenta; Feldman, Rodolfo Nestor; Gatti, Vanda Nora; Solis, Edita Amalia

2003-11-01

To investigate the presence of macrophages in human semen samples and the function they carry out in the seminal fluid. Their presence was studied in relation to spermatic morphology, percentage of spermatozoids with native DNA, and presence of antispermatic antibodies. The work was performed with semen samples from 31 unfertile males from 63 couples in which the "female factor" was ruled out as the cause of infertility. Sperm study according to WHO (1992) was carried out in all samples, in addition to: DNA study with acridine orange as fluorocrom, macrophage concentration by neutral red in a Neubauer camera, and detection of antispermatic antibodies with a mixed agglutination test (TAC II) (validated with Mar Screen-Fertility technologies). Sperm morphology was evaluated by Papanicolaou test. 19/31 selected sperm samples (61.3%) showed increased concentration of macrophages, 13 of them (41.9%) with denaturalized DNA, and 8 (25.8%) abnormal morphology. Six samples showed increased macrophage concentration and predominance of native DNA, whereas 11 samples showed increased macrophages and abnormal morphology. Among 18 (58.1%) samples showing antispermatic antibodies 14 (77.7%) had an increased concentration of macrophages. Statistical analysis resulted in a high correlation between macrophage concentration and increased percentage of spermatozoids with denaturalized DNA (p < 0.05). An increased concentration of macrophages is associated with the presence of antispermatic antibodies (p < 0.05). There was not evidence of significant association between concentration of macrophages and percentage of morphologically normal spermatozoids (p < 0.05). We can conclude that macrophages are present in human semen and participate in immunovigilance contributing to improve the seminal quality.

Cryopreservation of human blood for alkaline and Fpg-modified comet assay.

PubMed

Pu, Xinzhu; Wang, Zemin; Klaunig, James E

2016-01-01

The Comet assay is a reproducible and sensitive assay for the detection of DNA damage in eukaryotic cells and tissues. Incorporation of lesion specific, oxidative DNA damage repair enzymes (for example, Fpg, OGG1 and EndoIII) in the standard alkaline Comet assay procedure allows for the detection and measurement of oxidative DNA damage. The Comet assay using white blood cells (WBC) has proven useful in monitoring DNA damage from environmental agents in humans. However, it is often impractical to performance Comet assay immediately after blood sampling. Thus, storage of blood sample is required. In this study, we developed and tested a simple storage method for very small amount of whole blood for standard and Fpg-modified modified Comet assay. Whole blood was stored in RPMI 1640 media containing 10% FBS, 10% DMSO and 1 mM deferoxamine at a sample to media ratio of 1:50. Samples were stored at -20 °C and -80 °C for 1, 7, 14 and 28 days. Isolated lymphocytes from the same subjects were also stored under the same conditions for comparison. Direct DNA strand breakage and oxidative DNA damage in WBC and lymphocytes were analyzed using standard and Fpg-modified alkaline Comet assay and compared with freshly analyzed samples. No significant changes in either direct DNA strand breakage or oxidative DNA damage was seen in WBC and lymphocytes stored at -20 °C for 1 and 7 days compared to fresh samples. However, significant increases in both direct and oxidative DNA damage were seen in samples stored at -20 °C for 14 and 28 days. No changes in direct and oxidative DNA damage were observed in WBC and lymphocytes stored at -80 °C for up to 28 days. These results identified the proper storage conditions for storing whole blood or isolated lymphocytes to evaluate direct and oxidative DNA damage using standard and Fpg-modified alkaline Comet assay.

Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes.

PubMed

Cai, Xian-Quan; Yu, Hai-Qiong; Bai, Jian-Shan; Tang, Jian-Dong; Hu, Xu-Chu; Chen, Ding-Hu; Zhang, Ren-Li; Chen, Mu-Xin; Ai, Lin; Zhu, Xing-Quan

2012-03-01

Clonorchiasis caused by the oriental liver fluke Clonorchis sinensis is a fish-borne zoonosis endemic in a number of countries. This article describes the development of a TaqMan based real-time PCR assay for detection of C. sinensis DNA in human feces and in fishes. Primers targeting the first internal transcribed spacer (ITS-1) sequence of the fluke were highly specific for C. sinensis, as evidenced by the negative amplification of closely related trematodes in the test with the exception of Opisthorchis viverrini. The detection limit of the assay was 1pg of purified genomic DNA, 5EPG (eggs per gram feces) or one metacercaria per gram fish filet. The assay was evaluated by testing 22 human fecal samples and 37 fish tissues microscopically determined beforehand, and the PCR results were highly in agreement with the microscopic results. This real-time PCR assay provides a useful tool for the sensitive detection of C. sinensis DNA in human stool and aquatic samples in China and other endemic countries where O. viverrini and Opisthorchis felineus are absent. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Prevalence of human cell material: DNA and RNA profiling of public and private objects and after activity scenarios.

PubMed

van den Berge, M; Ozcanhan, G; Zijlstra, S; Lindenbergh, A; Sijen, T

2016-03-01

Especially when minute evidentiary traces are analysed, background cell material unrelated to the crime may contribute to detectable levels in the genetic analyses. To gain understanding on the composition of human cell material residing on surfaces contributing to background traces, we performed DNA and mRNA profiling on samplings of various items. Samples were selected by considering events contributing to cell material deposits in exemplary activities (e.g. dragging a person by the trouser ankles), and can be grouped as public objects, private samples, transfer-related samples and washing machine experiments. Results show that high DNA yields do not necessarily relate to an increased number of contributors or to the detection of other cell types than skin. Background cellular material may be found on any type of public or private item. When a major contributor can be deduced in DNA profiles from private items, this can be a different person than the owner of the item. Also when a specific activity is performed and the areas of physical contact are analysed, the "perpetrator" does not necessarily represent the major contributor in the STR profile. Washing machine experiments show that transfer and persistence during laundry is limited for DNA and cell type dependent for RNA. Skin conditions such as the presence of sebum or sweat can promote DNA transfer. Results of this study, which encompasses 549 samples, increase our understanding regarding the prevalence of human cell material in background and activity scenarios. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

DNA: The Strand that Connects Us All

ScienceCinema

Kaplan, Matt [Univ. of Arizona, Tucson, AZ (United States). Genetics Core Facility

2018-04-26

Learn how the methods and discoveries of human population genetics are applied for personal genealogical reconstruction and anthropological testing. Dr. Kaplan starts with a short general review of human genetics and the biology behind this form of DNA testing. He looks at how DNA testing is performed and how samples are processed in the University of Arizona laboratory. He also examines examples of personal genealogical results from Family Tree DNA and personal anthropological results from the Genographic Project. Finally, he describes the newest project in the UA laboratory, the DNA Shoah Project.

Human papillomavirus DNA detected in breast milk.

PubMed

Sarkola, Marja; Rintala, Marjut; Grénman, Seija; Syrjänen, Stina

2008-06-01

Human papillomavirus (HPV) testing of 223 breast milk samples 3 days postpartum was performed with polymerase chain reaction, hybridization, and sequencing. HPV-16-DNA was detected in 4.0% of the samples. HPV carriage in the breast milk was not correlated with mother's oral or cervical HPV-status or the demographic data. Oral HPV-infection of the spouse at month 6 and 12 postpartum was statistically significantly associated with HPV carriage in the breast milk.

High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA.

PubMed

Lundblom, Klara; Macharia, Alex; Lebbad, Marianne; Mohammed, Adan; Färnert, Anna

2011-08-08

Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome. High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene. High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection. High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots.

Previous Estimates of Mitochondrial DNA Mutation Level Variance Did Not Account for Sampling Error: Comparing the mtDNA Genetic Bottleneck in Mice and Humans

PubMed Central

Wonnapinij, Passorn; Chinnery, Patrick F.; Samuels, David C.

2010-01-01

In cases of inherited pathogenic mitochondrial DNA (mtDNA) mutations, a mother and her offspring generally have large and seemingly random differences in the amount of mutated mtDNA that they carry. Comparisons of measured mtDNA mutation level variance values have become an important issue in determining the mechanisms that cause these large random shifts in mutation level. These variance measurements have been made with samples of quite modest size, which should be a source of concern because higher-order statistics, such as variance, are poorly estimated from small sample sizes. We have developed an analysis of the standard error of variance from a sample of size n, and we have defined error bars for variance measurements based on this standard error. We calculate variance error bars for several published sets of measurements of mtDNA mutation level variance and show how the addition of the error bars alters the interpretation of these experimental results. We compare variance measurements from human clinical data and from mouse models and show that the mutation level variance is clearly higher in the human data than it is in the mouse models at both the primary oocyte and offspring stages of inheritance. We discuss how the standard error of variance can be used in the design of experiments measuring mtDNA mutation level variance. Our results show that variance measurements based on fewer than 20 measurements are generally unreliable and ideally more than 50 measurements are required to reliably compare variances with less than a 2-fold difference. PMID:20362273

Genetic signatures of ecological diversity along an urbanization gradient.

PubMed

Kelly, Ryan P; O'Donnell, James L; Lowell, Natalie C; Shelton, Andrew O; Samhouri, Jameal F; Hennessey, Shannon M; Feist, Blake E; Williams, Gregory D

2016-01-01

Despite decades of work in environmental science and ecology, estimating human influences on ecosystems remains challenging. This is partly due to complex chains of causation among ecosystem elements, exacerbated by the difficulty of collecting biological data at sufficient spatial, temporal, and taxonomic scales. Here, we demonstrate the utility of environmental DNA (eDNA) for quantifying associations between human land use and changes in an adjacent ecosystem. We analyze metazoan eDNA sequences from water sampled in nearshore marine eelgrass communities and assess the relationship between these ecological communities and the degree of urbanization in the surrounding watershed. Counter to conventional wisdom, we find strongly increasing richness and decreasing beta diversity with greater urbanization, and similar trends in the diversity of life histories with urbanization. We also find evidence that urbanization influences nearshore communities at local (hundreds of meters) rather than regional (tens of km) scales. Given that different survey methods sample different components of an ecosystem, we then discuss the advantages of eDNA-which we use here to detect hundreds of taxa simultaneously-as a complement to traditional ecological sampling, particularly in the context of broad ecological assessments where exhaustive manual sampling is impractical. Genetic data are a powerful means of uncovering human-ecosystem interactions that might otherwise remain hidden; nevertheless, no sampling method reveals the whole of a biological community.

Comparative Diagnosis of Human Bocavirus 1 Respiratory Infection With Messenger RNA Reverse-Transcription Polymerase Chain Reaction (PCR), DNA Quantitative PCR, and Serology.

PubMed

Xu, Man; Arku, Benedict; Jartti, Tuomas; Koskinen, Janne; Peltola, Ville; Hedman, Klaus; Söderlund-Venermo, Maria

2017-05-15

Human bocavirus (HBoV) 1 can cause life-threatening respiratory tract infection in children. Diagnosing acute HBoV1 infection is challenging owing to long-term airway persistence. We assessed whether messenger RNA (mRNA) detection would correlate better than DNA detection with acute HBoV1 infection. Paired serum samples from 121 children with acute wheezing were analyzed by means of serology. Quantitative polymerase chain reaction (PCR) and reverse-transcription (RT) PCR were applied to nasopharyngeal swab (NPS) samples from all acutely HBoV1-infected children and from controls with nonacute infection. By serology, 16 of 121 children (13.2%) had acute HBoV1 infection, all of whom had HBoV1 DNA in NPS samples, and 12 of 16 (75%) had HBoV1 mRNA. Among 25 children with nondiagnostic results, 6 had HBoV1 DNA in NPS samples, and 1 had mRNA. All 13 mRNA-positive samples exhibited high DNA loads (≥106 copies/mL). No mRNA persisted for 2 weeks, whereas HBoV1 DNA persisted for 2 months in 4 children; 1 year later all 15 samples were DNA negative. Compared with serology, DNA PCR had high clinical sensitivity (100%) but, because of viral persistence, low specificity (76%). In contrast, mRNA RT-PCR had low clinical sensitivity (75%) but high specificity (96%). A combination of HBoV1 serology and nasopharyngeal DNA quantitative PCR and mRNA RT-PCR should be used for accurate diagnosis of HBoV1 infection. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

A Fast Solution to NGS Library Prep with Low Nanogram DNA Input

PubMed Central

Liu, Pingfang; Lohman, Gregory J.S.; Cantor, Eric; Langhorst, Bradley W.; Yigit, Erbay; Apone, Lynne M.; Munafo, Daniela B.; Stewart, Fiona J.; Evans, Thomas C.; Nichols, Nicole; Dimalanta, Eileen T.; Davis, Theodore B.; Sumner, Christine

2013-01-01

Next Generation Sequencing (NGS) has significantly impacted human genetics, enabling a comprehensive characterization of the human genome as well as a better understanding of many genomic abnormalities. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized medicine a reality in the foreseeable future. To date, library construction with clinical samples has been a challenge, primarily due to the limited quantities of sample DNA available. Our objective here was to overcome this challenge by developing NEBNext® Ultra DNA Library Prep Kit, a fast library preparation method. Specifically, we streamlined the workflow utilizing novel NEBNext reagents and adaptors, including a new DNA polymerase that has been optimized to minimize GC bias. As a result of this work, we have developed a simple method for library construction from an amount of DNA as low as 5 ng, which can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms.

Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences.

PubMed

Wagner Mackenzie, Brett; Waite, David W; Taylor, Michael W

2015-01-01

The human gut contains dense and diverse microbial communities which have profound influences on human health. Gaining meaningful insights into these communities requires provision of high quality microbial nucleic acids from human fecal samples, as well as an understanding of the sources of variation and their impacts on the experimental model. We present here a systematic analysis of commonly used microbial DNA extraction methods, and identify significant sources of variation. Five extraction methods (Human Microbiome Project protocol, MoBio PowerSoil DNA Isolation Kit, QIAamp DNA Stool Mini Kit, ZR Fecal DNA MiniPrep, phenol:chloroform-based DNA isolation) were evaluated based on the following criteria: DNA yield, quality and integrity, and microbial community structure based on Illumina amplicon sequencing of the V4 region of bacterial and archaeal 16S rRNA genes. Our results indicate that the largest portion of variation within the model was attributed to differences between subjects (biological variation), with a smaller proportion of variation associated with DNA extraction method (technical variation) and intra-subject variation. A comprehensive understanding of the potential impact of technical variation on the human gut microbiota will help limit preventable bias, enabling more accurate diversity estimates.

Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer.

PubMed

Diede, Scott J; Yao, Zizhen; Keyes, C Chip; Tyler, Ashlee E; Dey, Joyoti; Hackett, Christopher S; Elsaesser, Katrina; Kemp, Christopher J; Neiman, Paul E; Weiss, William A; Olson, James M; Tapscott, Stephen J

2013-12-01

Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development.

Enhanced genetic analysis of single human bioparticles recovered by simplified micromanipulation from forensic 'touch DNA' evidence.

PubMed

Farash, Katherine; Hanson, Erin K; Ballantyne, Jack

2015-03-09

DNA profiles can be obtained from 'touch DNA' evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a 'blind-swabbing' approach will co-sample cellular material from the different individuals, even if the individuals' cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim's DNA may be found in significant excess thus masking any potential perpetrator's DNA. In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, 'smart analysis' method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 µl microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., "clumps") bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.

Electrospray ionization-tandem mass spectrometry and 32P-postlabeling analyses of tamoxifen-DNA adducts in humans.

PubMed

Beland, Frederick A; Churchwell, Mona I; Doerge, Daniel R; Parkin, Daniel R; Malejka-Giganti, Danuta; Hewer, Alan; Phillips, David H; Carmichael, Paul L; Gamboa da Costa, Gonçalo; Marques, M Matilde

2004-07-21

Although the nonsteroidal antiestrogen tamoxifen is used as an adjuvant chemotherapeutic agent to treat hormone-dependent breast cancer and as a chemopreventive agent in women with elevated risk of breast cancer, it has also been reported to increase the risk of endometrial cancer. Reports of low levels of tamoxifen-DNA adducts in human endometrial tissue have suggested that tamoxifen induces endometrial cancer by a genotoxic mechanism. However, these findings have been controversial. We used electrospray ionization-tandem mass spectrometry (ES-MS/MS) and 32P-postlabeling analyses to investigate the presence of tamoxifen-DNA adducts in human endometrial tissue. Endometrial DNA from eight tamoxifen-treated women and eight untreated women was hydrolyzed to nucleosides and assayed for (E)-alpha-(deoxyguanosin-N2-yl)-tamoxifen (dG-Tam) and (E)-alpha-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (dG-desMeTam), the two major tamoxifen-DNA adducts that have been reported to be present in humans and/or experimental animals treated with tamoxifen, using on-line sample preparation coupled with high-performance liquid chromatography (HPLC) and ES-MS/MS. The same DNA samples were assayed for the presence of dG-Tam and dG-desMeTam by (32)P-postlabeling methodology, using two different DNA digestion and labeling protocols, followed by both thin-layer chromatography and HPLC. We did not detect either tamoxifen-DNA adduct by HPLC-ES-MS/MS analyses (limits of detection for dG-Tam and dG-desMeTam were two adducts per 10(9) nucleotides and two adducts per 10(8) nucleotides, respectively) or by 32P-postlabeling analyses (limit of detection for both adducts was one adduct per 10(9) nucleotides) in any of the endometrial DNA samples. The initiation of endometrial cancer by tamoxifen is probably not due to a genotoxic mechanism involving the formation of dG-Tam or dG-desMeTam.

The Qiagen Investigator® Quantiplex HYres as an alternative kit for DNA quantification.

PubMed

Frégeau, Chantal J; Laurin, Nancy

2015-05-01

The Investigator® Quantiplex HYres kit was evaluated as a potential replacement for dual DNA quantification of casework samples. This kit was determined to be highly sensitive with a limit of quantification and limit of detection of 0.0049ng/μL and 0.0003ng/μL, respectively, for both human and male DNA, using full or half reaction volumes. It was also accurate in assessing the amount of male DNA present in 96 mock and actual casework male:female mixtures (various ratios) processed in this exercise. The close correlation between the male/human DNA ratios expressed in percentages derived from the Investigator® Quantiplex HYres quantification results and the male DNA proportion calculated in mixed AmpFlSTR® Profiler® Plus or AmpFlSTR® Identifiler® Plus profiles, using the Amelogenin Y peak and STR loci, allowed guidelines to be developed to facilitate decisions regarding when to submit samples to Y-STR rather than autosomal STR profiling. The internal control (IC) target was shown to be more sensitive to inhibitors compared to the human and male DNA targets included in the Investigator® Quantiplex HYres kit serving as a good quality assessor of DNA extracts. The new kit met our criteria of enhanced sensitivity, accuracy, consistency, reliability and robustness for casework DNA quantification. Crown Copyright © 2015. Published by Elsevier Ireland Ltd. All rights reserved.

Effects of microbial DNA on human DNA profiles generated using the PowerPlex® 16 HS system.

PubMed

Dembinski, Gina M; Picard, Christine J

2017-11-01

Most crime scenes are not sterile and therefore may be contaminated with environmental DNA, especially if a decomposing body is found. Collecting biological evidence from this individual will yield DNA samples mixed with microbial DNA. This also becomes important if postmortem swabs are collected from sexually assaulted victims. Although genotyping kits undergo validation tests, including bacterial screens, they do not account for the diverse microbial load during decomposition. We investigated the effect of spiking human DNA samples with known concentrations of DNA from 17 microbe species associated with decomposition on DNA profiles produced using the Promega PowerPlex ® HS system. Two species, Bacillus subtilis and Mycobacterium smegmatis, produced an extraneous allele at the TPOX locus. When repeated with the PowerPlex ® Fusion kit, the extra allele no longer amplified with these two species. This experiment demonstrates that caution should be exhibited if microbial load is high and the PowerPlex ® 16HS system is used. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Nonneutral mitochondrial DNA variation in humans and chimpanzees

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nachman, M.W.; Aquadro, C.F.; Brown, W.M.

1996-03-01

We sequenced the NADH dehydrogenase subunit 3 (ND3) gene from a sample of 61 humans, five common chimpanzees, and one gorilla to test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution. Within humans and within chimpanzees, the ratio of replacement to silent nucleotide substitutions was higher than observed in comparisons between species, contrary to neutral expectations. To test the generality of this result, we reanalyzed published human RFLP data from the entire mitochondrial genome. Gains of restriction sites relative to a known human mtDNA sequence were used to infer unambiguous nucleotide substitutions.more » We also compared the complete mtDNA sequences of three humans. Both the RFLP data and the sequence data reveal a higher ratio of replacement to silent nucleotide substitutions within humans than is seen between species. This pattern is observed at most or all human mitochondrial genes and is inconsistent with a strictly neutral model. These data suggest that many mitochondrial protein polymorphisms are slightly deleterious, consistent with studies of human mitochondrial diseases. 59 refs., 2 figs., 8 tabs.« less

High-speed shaking of frozen blood clots for extraction of human and malaria parasite DNA

PubMed Central

2011-01-01

Background Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome. Methods High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene. Results High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection. Conclusions High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots. PMID:21824391

ISFG: recommendations regarding the use of non-human (animal) DNA in forensic genetic investigations.

PubMed

Linacre, A; Gusmão, L; Hecht, W; Hellmann, A P; Mayr, W R; Parson, W; Prinz, M; Schneider, P M; Morling, N

2011-11-01

The use of non-human DNA typing in forensic science investigations, and specifically that from animal DNA, is ever increasing. The term animal DNA in this document refers to animal species encountered in a forensic science examination but does not include human DNA. Non-human DNA may either be: the trade and possession of a species, or products derived from a species, which is contrary to legislation; as evidence where the crime is against a person or property; instances of animal cruelty; or where the animal is the offender. The first instance is addressed by determining the species present, and the other scenarios can often be addressed by assigning a DNA sample to a particular individual organism. Currently there is little standardization of methodologies used in the forensic analysis of animal DNA or in reporting styles. The recommendations in this document relate specifically to animal DNA that is integral to a forensic science investigation and are not relevant to the breeding of animals for commercial purposes. This DNA commission was formed out of discussions at the International Society for Forensic Genetics 23rd Congress in Buenos Aires to outline recommendations on the use of non-human DNA in a forensic science investigation. Due to the scope of non-human DNA typing that is possible, the remit of this commission is confined to animal DNA typing only. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples.

PubMed

Besuschio, Susana A; Llano Murcia, Mónica; Benatar, Alejandro F; Monnerat, Severine; Cruz, Israel; Picado, Albert; Curto, María de Los Ángeles; Kubota, Yutaka; Wehrendt, Diana P; Pavia, Paula; Mori, Yasuyoshi; Puerta, Concepción; Ndung'u, Joseph M; Schijman, Alejandro G

2017-07-01

This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after "Boil & Spin" rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2 fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10-1 fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2 parasite equivalents/mL in spiked EDTA blood and 1x10-1 par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2 par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10-2 par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T.cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response.

Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples

PubMed Central

Besuschio, Susana A.; Llano Murcia, Mónica; Benatar, Alejandro F.; Monnerat, Severine; Cruz, Israel; Picado, Albert; Curto, María de los Ángeles; Kubota, Yutaka; Wehrendt, Diana P.; Pavia, Paula; Mori, Yasuyoshi; Puerta, Concepción; Ndung'u, Joseph M.

2017-01-01

This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T.cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after “Boil & Spin” rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10-2 fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10−1 fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10-2 parasite equivalents/mL in spiked EDTA blood and 1x10-1 par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10-2 par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10-2 par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T.cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response. PMID:28727723

Analysis of cellular response by exposure to acute or chronic radiation in human lymphoblastoid TK-6 cells

NASA Astrophysics Data System (ADS)

Ohnishi, T.; Yasumoto, J.; Takahashi, A.; Ohnishi, K.

To clarify the biological effects of low-dose rate radiation on human health for long-term stay in space, we analyzed the induction of apoptosis and apoptosis-related gene expression after irradiation with different dose-rate in human lymphoblastoid TK-6 cells harboring wild-type p53 gene. We irradiated TK-6 cells by X-ray at 1.5 Gy (1 Gy/min) and then sampled at 25 hr after culturing. We also irradiated by gamma-ray at 1.5 Gy (1 mGy/min) and then sampled immediately or 25 hr after irradiation. For DNA ladder analysis, we extracted DNA from these samples and electrophoresed with 2% agarose gel. In addition, we extracted mRNA from these samples for DNA-array analysis. mRNA from non-irradiated cells was used as a control. After labeling the cDNA against mRNA with [α -33P]-dCTP and hybridizing onto DNA array (Human Apoptosis Expression Array, R&D Systems), we scanned the profiles of the spots by a phosphorimager (BAS5000, FUJI FILM) and calculated using a NIH Image program. The data of each DNA-array were normalized with eight kinds of house keeping genes. We analyzed the expression level of apoptosis-related genes such as p53-related, Bcl-2 family, Caspase family and Fas-related genes. DNA ladders were obviously detected in the cells exposed to a high dose-rate radiation. We detected the induction of the gene expression of apoptosis-promotive genes. In contrast, almost no apoptosis was observed in the cells exposed to the chronic radiation at a low dose-rate. In addition, we detected the induction of the gene expression of apoptosis-suppressive genes as compared with apoptosis promotive-genes immediately after chronic irradiation. These results lead the importance of biological meaning of exposure to radiation at low dose-rate from an aspect of carcinogenesis. Finally, the effects of chronic irradiation become a highly important issue in space radiation biology for human health.

The currently used commercial DNA-extraction methods give different results of clostridial and actinobacterial populations derived from human fecal samples.

PubMed

Maukonen, Johanna; Simões, Catarina; Saarela, Maria

2012-03-01

Recently several human health-related microbiota studies have had partly contradictory results. As some differences may be explained by methodologies applied, we evaluated how different storage conditions and commonly used DNA-extraction kits affect bacterial composition, diversity, and numbers of human fecal microbiota. According to our results, the DNA-extraction did not affect the diversity, composition, or quantity of Bacteroides spp., whereas after a week's storage at -20 °C, the numbers of Bacteroides spp. were 1.6-2.5 log units lower (P < 0.05). Furthermore, the numbers of predominant bacteria, Eubacterium rectale (Erec)-group, Clostridium leptum group, bifidobacteria, and Atopobium group were 0.5-4 log units higher (P < 0.05) after mechanical DNA-extraction as detected with qPCR, regardless of storage. Furthermore, the bacterial composition of Erec-group differed significantly after different DNA-extractions; after enzymatic DNA-extraction, the most prevalent genera detected were Roseburia (39% of clones) and Coprococcus (10%), whereas after mechanical DNA-extraction, the most prevalent genera were Blautia (30%), Coprococcus (13%), and Dorea (10%). According to our results, rigorous mechanical lysis enables detection of higher bacterial numbers and diversity from human fecal samples. As it was shown that the results of clostridial and actinobacterial populations are highly dependent on the DNA-extraction methods applied, the use of different DNA-extraction protocols may explain the contradictory results previously obtained. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Atomic force microscope observation of branching in single transcript molecules derived from human cardiac muscle

NASA Astrophysics Data System (ADS)

Reed, Jason; Hsueh, Carlin; Mishra, Bud; Gimzewski, James K.

2008-09-01

We have used an atomic force microscope to examine a clinically derived sample of single-molecule gene transcripts, in the form of double-stranded cDNA, (c: complementary) obtained from human cardiac muscle without the use of polymerase chain reaction (PCR) amplification. We observed a log-normal distribution of transcript sizes, with most molecules being in the range of 0.4-7.0 kilobase pairs (kb) or 130-2300 nm in contour length, in accordance with the expected distribution of mRNA (m: messenger) sizes in mammalian cells. We observed novel branching structures not previously known to exist in cDNA, and which could have profound negative effects on traditional analysis of cDNA samples through cloning, PCR and DNA sequencing.

The presence of human papillomavirus in semen does not affect the integrity of sperm DNA.

PubMed

Cortés-Gutiérrez, E I; Dávila-Rodríguez, M I; Fernández, J L; de la O-Pérez, L O; Garza-Flores, M E; Eguren-Garza, R; Gosálvez, J

2017-12-01

It remains unknown whether human papillomaviruses (HPVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen samples of 22 normozoospermic patients undergoing infertility treatment, nine fertile donors and seven fertile men with a risk of HPV infection (genital warts or condylomas) were included in the study. The samples were examined by an INNO-LiPA test PCR-based reverse hybridisation array that identifies 28 types of HPVs as simple or multiple infections. Sperm DNA integrity was determined by sperm chromatin dispersion assay (SCD). Our preliminary findings demonstrate an increase in HPV infection in infertile men with respect to fertile men. However, the sperm DNA fragmentation index was not increased in semen containing these viruses. © 2017 Blackwell Verlag GmbH.

Direct-to-PCR tissue preservation for DNA profiling.

PubMed

Sorensen, Amy; Berry, Clare; Bruce, David; Gahan, Michelle Elizabeth; Hughes-Stamm, Sheree; McNevin, Dennis

2016-05-01

Disaster victim identification (DVI) often occurs in remote locations with extremes of temperatures and humidities. Access to mortuary facilities and refrigeration are not always available. An effective and robust DNA sampling and preservation procedure would increase the probability of successful DNA profiling and allow faster repatriation of bodies and body parts. If the act of tissue preservation also released DNA into solution, ready for polymerase chain reaction (PCR), the DVI process could be further streamlined. In this study, we explored the possibility of obtaining DNA profiles without DNA extraction, by adding aliquots of preservative solutions surrounding fresh human muscle and decomposing human muscle and skin tissue samples directly to PCR. The preservatives consisted of two custom preparations and two proprietary solutions. The custom preparations were a salt-saturated solution of dimethyl sulfoxide (DMSO) with ethylenediaminetetraacetic (EDTA) and TENT buffer (Tris, EDTA, NaCl, Tween 20). The proprietary preservatives were DNAgard (Biomatrica(®)) and Tissue Stabilising Kit (DNA Genotek). We obtained full PowerPlex(®) 21 (Promega) and GlobalFiler(®) (Life Technologies) DNA profiles from fresh and decomposed tissue preserved at 35 °C for up to 28 days for all four preservatives. The preservative aliquots removed from the fresh muscle tissue samples had been stored at -80 °C for 4 years, indicating that long-term archival does not diminish the probability of successful DNA typing. Rather, storage at -80 °C seems to reduce PCR inhibition.

Persistence of human immunodeficiency virus type 1 subtype B DNA in dried-blood samples on FTA filter paper.

PubMed

Li, Chung-Chen; Beck, Ingrid A; Seidel, Kristy D; Frenkel, Lisa M

2004-08-01

The stability of human immunodeficiency virus type 1 (HIV-1) DNA in whole blood collected on filter paper (FTA Card) was evaluated. After >4 years of storage at room temperature in the dark our qualitative assay detected virus at a rate similar to that of our initial test (58 of 60, 97%; P = 0.16), suggesting long-term HIV-1 DNA stability.

Persistence of Human Immunodeficiency Virus Type 1 Subtype B DNA in Dried-Blood Samples on FTA Filter Paper

PubMed Central

Li, Chung-Chen; Beck, Ingrid A.; Seidel, Kristy D.; Frenkel, Lisa M.

2004-01-01

The stability of human immunodeficiency virus type 1 (HIV-1) DNA in whole blood collected on filter paper (FTA Card) was evaluated. After >4 years of storage at room temperature in the dark our qualitative assay detected virus at a rate similar to that of our initial test (58 of 60, 97%; P = 0.16), suggesting long-term HIV-1 DNA stability. PMID:15297546

The forensiX evidence collection tube and its impact on DNA preservation and recovery.

PubMed

Garvin, Alex M; Holzinger, Ralf; Berner, Florian; Krebs, Walter; Hostettler, Bernhard; Lardi, Elges; Hertli, Christian; Quartermaine, Roy; Stamm, Christoph

2013-01-01

Biological samples are vulnerable to degradation from the time they are collected until they are analysed at the laboratory. Biological contaminants, such as bacteria, fungi, and enzymes, as well as environmental factors, such as sunlight, heat, and humidity, can increase the rate of DNA degradation. Currently, DNA samples are normally dried or frozen to limit their degradation prior to their arrival at the laboratory. In this study, the effect of the sample drying rate on DNA preservation was investigated, as well as a comparison between drying and freezing methods. The drying performances of two commercially available DNA collection tools (swab and drying tube) with different drying rates were evaluated. The swabs were used to collect human saliva, placed into the drying tubes, and stored in a controlled environment at 25°C and 60% relative humidity, or frozen at -20°C, for 2 weeks. Swabs that were stored in fast sample drying tubes yielded 95% recoverable DNA, whereas swabs stored in tubes with slower sample drying rates yielded only 12% recoverable DNA; saliva stored in a microtube at -20°C was used as a control. Thus, DNA sampling tools that offer rapid drying can significantly improve the preservation of DNA collected on a swab, increasing the quantity of DNA available for subsequent analysis.

Detection of a Diverse Marine Fish Fauna Using Environmental DNA from Seawater Samples

PubMed Central

Iversen, Lars Lønsmann; Møller, Peter Rask; Rasmussen, Morten; Willerslev, Eske

2012-01-01

Marine ecosystems worldwide are under threat with many fish species and populations suffering from human over-exploitation. This is greatly impacting global biodiversity, economy and human health. Intriguingly, marine fish are largely surveyed using selective and invasive methods, which are mostly limited to commercial species, and restricted to particular areas with favourable conditions. Furthermore, misidentification of species represents a major problem. Here, we investigate the potential of using metabarcoding of environmental DNA (eDNA) obtained directly from seawater samples to account for marine fish biodiversity. This eDNA approach has recently been used successfully in freshwater environments, but never in marine settings. We isolate eDNA from ½-litre seawater samples collected in a temperate marine ecosystem in Denmark. Using next-generation DNA sequencing of PCR amplicons, we obtain eDNA from 15 different fish species, including both important consumption species, as well as species rarely or never recorded by conventional monitoring. We also detect eDNA from a rare vagrant species in the area; European pilchard (Sardina pilchardus). Additionally, we detect four bird species. Records in national databases confirmed the occurrence of all detected species. To investigate the efficiency of the eDNA approach, we compared its performance with 9 methods conventionally used in marine fish surveys. Promisingly, eDNA covered the fish diversity better than or equal to any of the applied conventional methods. Our study demonstrates that even small samples of seawater contain eDNA from a wide range of local fish species. Finally, in order to examine the potential dispersal of eDNA in oceans, we performed an experiment addressing eDNA degradation in seawater, which shows that even small (100-bp) eDNA fragments degrades beyond detectability within days. Although further studies are needed to validate the eDNA approach in varying environmental conditions, our findings provide a strong proof-of-concept with great perspectives for future monitoring of marine biodiversity and resources. PMID:22952584

Detection of a diverse marine fish fauna using environmental DNA from seawater samples.

PubMed

Thomsen, Philip Francis; Kielgast, Jos; Iversen, Lars Lønsmann; Møller, Peter Rask; Rasmussen, Morten; Willerslev, Eske

2012-01-01

Marine ecosystems worldwide are under threat with many fish species and populations suffering from human over-exploitation. This is greatly impacting global biodiversity, economy and human health. Intriguingly, marine fish are largely surveyed using selective and invasive methods, which are mostly limited to commercial species, and restricted to particular areas with favourable conditions. Furthermore, misidentification of species represents a major problem. Here, we investigate the potential of using metabarcoding of environmental DNA (eDNA) obtained directly from seawater samples to account for marine fish biodiversity. This eDNA approach has recently been used successfully in freshwater environments, but never in marine settings. We isolate eDNA from ½-litre seawater samples collected in a temperate marine ecosystem in Denmark. Using next-generation DNA sequencing of PCR amplicons, we obtain eDNA from 15 different fish species, including both important consumption species, as well as species rarely or never recorded by conventional monitoring. We also detect eDNA from a rare vagrant species in the area; European pilchard (Sardina pilchardus). Additionally, we detect four bird species. Records in national databases confirmed the occurrence of all detected species. To investigate the efficiency of the eDNA approach, we compared its performance with 9 methods conventionally used in marine fish surveys. Promisingly, eDNA covered the fish diversity better than or equal to any of the applied conventional methods. Our study demonstrates that even small samples of seawater contain eDNA from a wide range of local fish species. Finally, in order to examine the potential dispersal of eDNA in oceans, we performed an experiment addressing eDNA degradation in seawater, which shows that even small (100-bp) eDNA fragments degrades beyond detectability within days. Although further studies are needed to validate the eDNA approach in varying environmental conditions, our findings provide a strong proof-of-concept with great perspectives for future monitoring of marine biodiversity and resources.

Evidence of authentic DNA from Danish Viking Age skeletons untouched by humans for 1,000 years.

PubMed

Melchior, Linea; Kivisild, Toomas; Lynnerup, Niels; Dissing, Jørgen

2008-05-28

Given the relative abundance of modern human DNA and the inherent impossibility for incontestable proof of authenticity, results obtained on ancient human DNA have often been questioned. The widely accepted rules regarding ancient DNA work mainly affect laboratory procedures, however, pre-laboratory contamination occurring during excavation and archaeological-/anthropological handling of human remains as well as rapid degradation of authentic DNA after excavation are major obstacles. We avoided some of these obstacles by analyzing DNA from ten Viking Age subjects that at the time of sampling were untouched by humans for 1,000 years. We removed teeth from the subjects prior to handling by archaeologists and anthropologists using protective equipment. An additional tooth was removed after standard archaeological and anthropological handling. All pre-PCR work was carried out in a "clean- laboratory" dedicated solely to ancient DNA work. Mitochondrial DNA was extracted and overlapping fragments spanning the HVR-1 region as well as diagnostic sites in the coding region were PCR amplified, cloned and sequenced. Consistent results were obtained with the "unhandled" teeth and there was no indication of contamination, while the latter was the case with half of the "handled" teeth. The results allowed the unequivocal assignment of a specific haplotype to each of the subjects, all haplotypes being compatible in their character states with a phylogenetic tree drawn from present day European populations. Several of the haplotypes are either infrequent or have not been observed in modern Scandinavians. The observation of haplogroup I in the present study (<2% in modern Scandinavians) supports our previous findings of a pronounced frequency of this haplogroup in Viking and Iron Age Danes. The present work provides further evidence that retrieval of ancient human DNA is a possible task provided adequate precautions are taken and well-considered sampling is applied.

Evidence of Authentic DNA from Danish Viking Age Skeletons Untouched by Humans for 1,000 Years

PubMed Central

Melchior, Linea; Kivisild, Toomas; Lynnerup, Niels; Dissing, Jørgen

2008-01-01

Background Given the relative abundance of modern human DNA and the inherent impossibility for incontestable proof of authenticity, results obtained on ancient human DNA have often been questioned. The widely accepted rules regarding ancient DNA work mainly affect laboratory procedures, however, pre-laboratory contamination occurring during excavation and archaeological-/anthropological handling of human remains as well as rapid degradation of authentic DNA after excavation are major obstacles. Methodology/Principal Findings We avoided some of these obstacles by analyzing DNA from ten Viking Age subjects that at the time of sampling were untouched by humans for 1,000 years. We removed teeth from the subjects prior to handling by archaeologists and anthropologists using protective equipment. An additional tooth was removed after standard archaeological and anthropological handling. All pre-PCR work was carried out in a “clean- laboratory” dedicated solely to ancient DNA work. Mitochondrial DNA was extracted and overlapping fragments spanning the HVR-1 region as well as diagnostic sites in the coding region were PCR amplified, cloned and sequenced. Consistent results were obtained with the “unhandled” teeth and there was no indication of contamination, while the latter was the case with half of the “handled” teeth. The results allowed the unequivocal assignment of a specific haplotype to each of the subjects, all haplotypes being compatible in their character states with a phylogenetic tree drawn from present day European populations. Several of the haplotypes are either infrequent or have not been observed in modern Scandinavians. The observation of haplogroup I in the present study (<2% in modern Scandinavians) supports our previous findings of a pronounced frequency of this haplogroup in Viking and Iron Age Danes. Conclusion The present work provides further evidence that retrieval of ancient human DNA is a possible task provided adequate precautions are taken and well-considered sampling is applied. PMID:18509537

Multiplex picoliter-droplet digital PCR for quantitative assessment of DNA integrity in clinical samples.

PubMed

Didelot, Audrey; Kotsopoulos, Steve K; Lupo, Audrey; Pekin, Deniz; Li, Xinyu; Atochin, Ivan; Srinivasan, Preethi; Zhong, Qun; Olson, Jeff; Link, Darren R; Laurent-Puig, Pierre; Blons, Hélène; Hutchison, J Brian; Taly, Valerie

2013-05-01

Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet-based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA. Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues. One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants. The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples. © 2013 American Association for Clinical Chemistry.

Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility.

PubMed

Ollero, M; Gil-Guzman, E; Lopez, M C; Sharma, R K; Agarwal, A; Larson, K; Evenson, D; Thomas, A J; Alvarez, J G

2001-09-01

Reactive oxygen species (ROS)-induced damage of membrane phospholipids and DNA in human spermatozoa has been implicated in the pathogenesis of male infertility. In this study, variations in ROS production, DNA structure (as measured by the sperm chromatin structure assay) and lipid composition, were studied in human spermatozoa at different stages of maturation. Sperm subsets were isolated by discontinuous density gradient centrifugation of semen samples obtained from healthy donors and from infertility patients. DNA damage and ROS production were highest in immature spermatozoa with cytoplasmic retention and abnormal head morphology, and lowest in mature spermatozoa. Docosahexaenoic acid and sterol content were highest in immature germ cells and immature spermatozoa, and lowest in mature spermatozoa. The relative proportion of ROS-producing immature spermatozoa in the sample was directly correlated with DNA damage in mature spermatozoa, and inversely correlated with the recovery of motile spermatozoa. There was no correlation between DNA damage and sperm morphology in mature spermatozoa. The high levels of ROS production and DNA damage observed in immature spermatozoa may be indicative of derangements in the regulation of spermiogenesis. DNA damage in mature spermatozoa may be the result of oxidative damage by ROS-producing immature spermatozoa during sperm migration from the seminiferous tubules to the epididymis.

No Evidence of Human Papilloma Virus Infection in Basal Cell Carcinoma

PubMed Central

Nahidi, Yalda; Meibodi, Naser Tayyebi; Meshkat, Zahra; Esmaili, Habibollah; Jahanfakhr, Samaneh

2015-01-01

Background: Basal cell carcinoma (BCC) is the most common skin cancer among whites, and several risk factors have been discussed in itsdevelopment and progress. Detection of human papilloma virus (HPV) deoxyribonucleic acid (DNA) BCCs in some studies suggests that the virus may play a role in the pathogenesis of this disease. Several molecular studies showed conflicting reports. Aims: The purpose of this study was to investigate the association between HPV and BCC using polymerase chain reaction (PCR). Materials and Methods: HPV DNA detection was done for 42 paraffin-embedded tissue specimens of BCC and 42 normal skin samples around the lesions by PCR using GP5+/GP6+ primers. Results: HPV DNA was not found in any of the 42 samples of BCC, and only one normal skin sample around the lesions was positive for HPV DNA by PCR. Conclusion: In this study, no statistically significant difference was seen between the presence of HPV DNA in BCC and normal skin around the lesion, and HPV is not likely to have an important role in pathogenesis of BCC. PMID:26288402

The Iranian Human Mutation Gene Bank: a data and sample resource for worldwide collaborative genetics research.

PubMed

Najmabadi, Hossein; Neishabury, Maryam; Sahebjam, Farhad; Kahrizi, Kimia; Shafaghati, Yousef; Nikzat, Nushin; Jalalvand, Maryam; Aminy, Farahnaz; Hashemi, Susan Bany; Moghimi, Babak; Noorian, Ali Reza; Jannati, Ali; Mohammadi, Mehrdad; Javan, Khalil

2003-02-01

As Human Genome Project exploration continues, the necessity of having a broader spectrum of genomic DNA material from different nationalities to study various aspects of hereditary disease becomes more obvious. The existence of high genetic polymorphism within and between different communities in the world makes it necessary for the gene hunters to investigate many different populations. Iran, a large country with close to 66 million people, is a land of different nationalities, tribes, and religions that offers a highly heterogeneous gene pool to the genetics researcher. The purity of many different races in this country has been highly conserved by geographical borders and by an ancient culture that has always encouraged intrafamilial marriages. All these have created a population that is remarkably heterogeneous yet high in consanguinity rate. During the last five years of investigation we have established a DNA bank, the Iranian Human Mutation Gene Bank (www.IHMGB.com), which contains all genetic diseases studied in Iran that have the Mendelian mode of inheritance. Some of the samples are assigned to common or novel mutations and others belong to patients with clinical profiles associated with particular genetic diseases but undefined mutation. This bank stores samples of DNA from the patient and his/her first-degree relatives together with a comprehensive pedigree and clinical profile for each sample. To facilitate collaboration with other scientists around the world with the same interests, we decided to present our experimental projects online. This DNA bank provides opportunities for us to collaborate with scientists outside Iran. It offers a sample resource to research scientists around the world, at no charge, for the purpose of investigating the various aspects of genetic disorders from prenatal diagnosis to gene structure and function. It is strongly stressed that no commercial benefit is involved in the establishment of this DNA bank and the DNA samples are free of charge. However, to meet our goals and to respect ethical values, DNA samples can only be used under certain conditions stated in the User Consent Form. Copyright 2003 Wiley-Liss, Inc.

Plasmodium falciparum-like parasites infecting wild apes in southern Cameroon do not represent a recurrent source of human malaria

PubMed Central

Sundararaman, Sesh A.; Liu, Weimin; Keele, Brandon F.; Learn, Gerald H.; Bittinger, Kyle; Mouacha, Fatima; Ahuka-Mundeke, Steve; Manske, Magnus; Sherrill-Mix, Scott; Li, Yingying; Malenke, Jordan A.; Delaporte, Eric; Laurent, Christian; Mpoudi Ngole, Eitel; Kwiatkowski, Dominic P.; Shaw, George M.; Rayner, Julian C.; Peeters, Martine; Sharp, Paul M.; Bushman, Frederic D.; Hahn, Beatrice H.

2013-01-01

Wild-living chimpanzees and gorillas harbor a multitude of Plasmodium species, including six of the subgenus Laverania, one of which served as the progenitor of Plasmodium falciparum. Despite the magnitude of this reservoir, it is unknown whether apes represent a source of human infections. Here, we used Plasmodium species-specific PCR, single-genome amplification, and 454 sequencing to screen humans from remote areas of southern Cameroon for ape Laverania infections. Among 1,402 blood samples, we found 1,000 to be Plasmodium mitochondrial DNA (mtDNA) positive, all of which contained human parasites as determined by sequencing and/or restriction enzyme digestion. To exclude low-abundance infections, we subjected 514 of these samples to 454 sequencing, targeting a region of the mtDNA genome that distinguishes ape from human Laverania species. Using algorithms specifically developed to differentiate rare Plasmodium variants from 454-sequencing error, we identified single and mixed-species infections with P. falciparum, Plasmodium malariae, and/or Plasmodium ovale. However, none of the human samples contained ape Laverania parasites, including the gorilla precursor of P. falciparum. To characterize further the diversity of P. falciparum in Cameroon, we used single-genome amplification to amplify 3.4-kb mtDNA fragments from 229 infected humans. Phylogenetic analysis identified 62 new variants, all of which clustered with extant P. falciparum, providing further evidence that P. falciparum emerged following a single gorilla-to-human transmission. Thus, unlike Plasmodium knowlesi-infected macaques in southeast Asia, African apes harboring Laverania parasites do not seem to serve as a recurrent source of human malaria, a finding of import to ongoing control and eradication measures. PMID:23569255

Quantitation of exposure to benzo[a]pyrene with monoclonal antibodies.

PubMed Central

Santella, R M; Hsieh, L L; Lin, C D; Viet, S; Weinstein, I B

1985-01-01

It is now possible to quantitate carcinogen adducts on DNA by highly sensitive immunoassays. These techniques are particularly useful for screening human populations for exposure to potential environmental carcinogens. We have developed a panel of monoclonal antibodies that react with benzo(a)pyrene (BP) modified DNA to be used in an enzyme linked immunoassay (ELISA) to quantitate adduct levels of both human and animal samples. BALBc/Cr mice were immunized with either DNA modified by 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo(a)pyrene (BPDE-I-DNA) complexed electrostatically to methylated bovine serum albumin or with BPDE-I-modified guanosine conjugated with bovine serum albumin (BPDE-I-G-BSA). Four stable clones were produced from the spleen cells of animals immunized with BPDE-I-DNA and one from BPDE-I-G-BSA immunized animals. All antibodies were shown to be highly specific for BPDE-I-DNA and did not crossreact with nonmodified DNA or with N-2-acetylaminofluorene or 1-aminopyrene modified DNA. The antibodies differed in their sensitivity to BPDE-II-DNA, BPDE-I-poly G, BPDE-I-tetraols and BPDE-I-dG. In general, all the antibodies showed the greatest affinity for their original antigen. Those generated against modified DNA showed highest reactivity against modified DNA while the one antibody generated against the monoadduct showed highest reactivity with the monoadduct. These antibodies are currently being used in a highly sensitive competitive ELISA to quantitate levels of BP-DNA adducts in various animal and human tissue samples. PMID:4085452

Detection of Coxiella burnetii DNA in Inhalable Airborne Dust Samples from Goat Farms after Mandatory Culling

PubMed Central

Hogerwerf, Lenny; Still, Kelly; Heederik, Dick; van Rotterdam, Bart; de Bruin, Arnout; Nielen, Mirjam; Wouters, Inge M.

2012-01-01

Coxiella burnetii is thought to infect humans primarily via airborne transmission. However, air measurements of C. burnetii are sparse. We detected C. burnetii DNA in inhalable and PM10 (particulate matter with an aerodynamic size of 10 μm or less) dust samples collected at three affected goat farms, demonstrating that low levels of C. burnetii DNA are present in inhalable size fractions. PMID:22582072

Diverse replication-associated protein encoding circular DNA viruses in guano samples of Central-Eastern European bats.

PubMed

Kemenesi, Gábor; Kurucz, Kornélia; Zana, Brigitta; Földes, Fanni; Urbán, Péter; Vlaschenko, Anton; Kravchenko, Kseniia; Budinski, Ivana; Szodoray-Parádi, Farkas; Bücs, Szilárd; Jére, Csaba; Csősz, István; Szodoray-Parádi, Abigél; Estók, Péter; Görföl, Tamás; Boldogh, Sándor; Jakab, Ferenc

2018-03-01

Circular replication-associated protein encoding single-stranded DNA (CRESS DNA) viruses are increasingly recognized worldwide in a variety of samples. Representative members include well-described veterinary pathogens with worldwide distribution, such as porcine circoviruses or beak and feather disease virus. In addition, numerous novel viruses belonging to the family Circoviridae with unverified pathogenic roles have been discovered in different human samples. Viruses of the family Genomoviridae have also been described as being highly abundant in different faecal and environmental samples, with case reports showing them to be suspected pathogens in human infections. In order to investigate the genetic diversity of these viruses in European bat populations, we tested guano samples from Georgia, Hungary, Romania, Serbia and Ukraine. This resulted in the detection of six novel members of the family Circoviridae and two novel members of the family Genomoviridae. Interestingly, a gemini-like virus, namely niminivirus, which was originally found in raw sewage samples in Nigeria, was also detected in our samples. We analyzed the nucleotide composition of members of the family Circoviridae to determine the possible host origins of these viruses. This study provides the first dataset on CRESS DNA viruses of European bats, and members of several novel viral species were discovered.

Assessment of Multiple Types of DNA Damage in Human Placentas from Smoking and Non-smoking Women in the Czech Republic

PubMed Central

Margaret Pratt, M.; King, Leon C.; Adams, Linda D.; John, Kaarthik; Sirajuddin, Paul; Olivero, Ofelia A.; Manchester, David K.; Sram, Radim J.; DeMarini, David M.; Poirier, Miriam C.

2010-01-01

Three classes of DNA damage were assessed in human placentas collected (in 2000-4) from 51 women living in the Teplice region of the Czech Republic, a mining area considered to have some of the worst environmental pollution in Europe in the 1980s. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were localized and semiquantified using immunohistochemistry (IHC) and the Automated Cellular Imaging System (ACIS). More generalized DNA damage was measured both by 32P-postlabeling and by abasic (AB) site analysis. Placenta stained with antiserum elicited against DNA modified with r7, t8-dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) revealed PAH-DNA adduct localization in nuclei of the cytotrophoblast (CT) cells and syncytiotrophoblast (ST) knots lining the chorionic villi. The highest levels of DNA damage, 49–312 PAH-DNA adducts/108 nucleotides, were found by IHC/ACIS in 14 immediately-fixed placenta samples. An additional 37 placenta samples were stored frozen before fixation and embedding, and because PAH-DNA adducts were largely undetectable in these samples, freezing was implicated in the loss of IHC signal. The same placentas (n = 37) contained 1.7 – 8.6 stable/bulky DNA adducts/108 nucleotides and 0.6 – 47.2 AB sites/105 nucleotides. For all methods there was no correlation among types of DNA damage and no difference in extent of DNA damage between smokers and non-smokers. Therefore, the data show that DNA from placentas obtained in Teplice contained multiple types of DNA damage, which likely arose from various environmental exposures. In addition, PAH-DNA adducts were present at high concentrations in the CT cells and ST knots of the chorionic villi. PMID:20839217

Assessment of multiple types of DNA damage in human placentas from smoking and nonsmoking women in the Czech Republic.

PubMed

Pratt, M Margaret; King, Leon C; Adams, Linda D; John, Kaarthik; Sirajuddin, Paul; Olivero, Ofelia A; Manchester, David K; Sram, Radim J; DeMarini, David M; Poirier, Miriam C

2011-01-01

Three classes of DNA damage were assessed in human placentas collected (2000-2004) from 51 women living in the Teplice region of the Czech Republic, a mining area considered to have some of the worst environmental pollution in Europe in the 1980s. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were localized and semiquantified using immunohistochemistry (IHC) and the Automated Cellular Imaging System (ACIS). More generalized DNA damage was measured both by (32)P-postlabeling and by abasic (AB) site analysis. Placenta stained with antiserum elicited against DNA modified with 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) revealed PAH-DNA adduct localization in nuclei of the cytotrophoblast (CT) cells and syncytiotrophoblast (ST) knots lining the chorionic villi. The highest levels of DNA damage, 49-312 PAH-DNA adducts/10(8) nucleotides, were found by IHC/ACIS in 14 immediately fixed placenta samples. An additional 37 placenta samples were stored frozen before fixation and embedding, and because PAH-DNA adducts were largely undetectable in these samples, freezing was implicated in the loss of IHC signal. The same placentas (n = 37) contained 1.7-8.6 stable/bulky DNA adducts/10(8) nucleotides and 0.6-47.2 AB sites/10(5) nucleotides. For all methods, there was no correlation among types of DNA damage and no difference in extent of DNA damage between smokers and nonsmokers. Therefore, the data show that DNA from placentas obtained in Teplice contained multiple types of DNA damage, which likely arose from various environmental exposures. In addition, PAH-DNA adducts were present at high concentrations in the CT cells and ST knots of the chorionic villi. Copyright © 2010 Wiley-Liss, Inc.

Prevalence of human herpes virus types 1-7 in the semen of men attending an infertility clinic and correlation with semen parameters.

PubMed

Neofytou, Eirini; Sourvinos, George; Asmarianaki, Maria; Spandidos, Demetrios A; Makrigiannakis, Antonios

2009-06-01

To determine the prevalence of herpes viruses in the semen of an asymptomatic male cohort with and without infertility problems and its association with altered semen parameters. A prospective randomized study. Medical school and IVF clinic. One hundred seventy-two male patients undergoing routine semen analysis: 80 with normal semen parameters (control group) and 92 with abnormal semen parameters. Semen samples were collected by masturbation. The DNA from the Herpesviridae family (herpes simplex virus 1 [HSV-1], herpes simplex virus 2 [HSV-2], Varicella zoster virus [VZV], Epstein-Barr virus [EBV], cytomegalovirus [CMV], human herpes virus type 6 [HHV-6], human herpes virus type 7 [HHV-7]) and routine semen parameters. Viral DNA was detected in 143/172 (83.1%) of the total samples for at least one herpes virus: HSV-1, 2.5%; VZV, 1.2%; EBV, 45%; CMV, 62.5%; HHV-6, 70%; HHV-7, 0% in the normal semen samples and HSV-1, 2.1%; VZV, 3.2%; EBV, 39.1%; CMV, 56.5%; HHV-6, 66.3%; HHV-7, 0% in the abnormal semen samples. No association was found between the presence of viral DNA and semen parameters. Interestingly, a statistical significance between leukocytospermia and the presence of EBV DNA was observed. The DNA of herpes viruses is frequently detected in the semen of asymptomatic fertile and infertile male patients. Further studies are required to investigate the role of herpes viruses in male factor infertility.

Ancient DNA Reveals Matrilineal Continuity in Present-Day Poland over the Last Two Millennia

PubMed Central

Juras, Anna; Dabert, Miroslawa; Kushniarevich, Alena; Malmström, Helena; Raghavan, Maanasa; Kosicki, Jakub Z.; Metspalu, Ene; Willerslev, Eske; Piontek, Janusz

2014-01-01

While numerous ancient human DNA datasets from across Europe have been published till date, modern-day Poland in particular, remains uninvestigated. Besides application in the reconstruction of continent-wide human history, data from this region would also contribute towards our understanding of the history of the Slavs, whose origin is hypothesized to be in East or Central Europe. Here, we present the first population-scale ancient human DNA study from the region of modern-day Poland by establishing mitochondrial DNA profiles for 23 samples dated to 200 BC – 500 AD (Roman Iron Age) and for 20 samples dated to 1000–1400 AD (Medieval Age). Our results show that mitochondrial DNA sequences from both periods belong to haplogroups that are characteristic of contemporary West Eurasia. Haplotype sharing analysis indicates that majority of the ancient haplotypes are widespread in some modern Europeans, including Poles. Notably, the Roman Iron Age samples share more rare haplotypes with Central and Northeast Europeans, whereas the Medieval Age samples share more rare haplotypes with East-Central and South-East Europeans, primarily Slavic populations. Our data demonstrates genetic continuity of certain matrilineages (H5a1 and N1a1a2) in the area of present-day Poland from at least the Roman Iron Age until present. As such, the maternal gene pool of present-day Poles, Czechs and Slovaks, categorized as Western Slavs, is likely to have descended from inhabitants of East-Central Europe during the Roman Iron Age. PMID:25337992

Development of procedures for the identification of human papilloma virus DNA fragments in laser plume

NASA Astrophysics Data System (ADS)

Woellmer, Wolfgang; Meder, Tom; Jappe, Uta; Gross, Gerd; Riethdorf, Sabine; Riethdorf, Lutz; Kuhler-Obbarius, Christina; Loening, Thomas

1996-01-01

For the investigation of laser plume for the existence of HPV DNA fragments, which possibly occur during laser treatment of virus infected tissue, human papillomas and condylomas were treated in vitro with the CO2-laser. For the sampling of the laser plume a new method for the trapping of the material was developed by use of water-soluble gelatine filters. These samples were analyzed with the polymerase chain reaction (PCR) technique, which was optimized in regard of the gelatine filters and the specific primers. Positive PCR results for HPV DNA fragments up to the size of a complete oncogene were obtained and are discussed regarding infectiousity.

International Study to Evaluate PCR Methods for Detection of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

PubMed Central

Schijman, Alejandro G.; Bisio, Margarita; Orellana, Liliana; Sued, Mariela; Duffy, Tomás; Mejia Jaramillo, Ana M.; Cura, Carolina; Auter, Frederic; Veron, Vincent; Qvarnstrom, Yvonne; Deborggraeve, Stijn; Hijar, Gisely; Zulantay, Inés; Lucero, Raúl Horacio; Velazquez, Elsa; Tellez, Tatiana; Sanchez Leon, Zunilda; Galvão, Lucia; Nolder, Debbie; Monje Rumi, María; Levi, José E.; Ramirez, Juan D.; Zorrilla, Pilar; Flores, María; Jercic, Maria I.; Crisante, Gladys; Añez, Néstor; De Castro, Ana M.; Gonzalez, Clara I.; Acosta Viana, Karla; Yachelini, Pedro; Torrico, Faustino; Robello, Carlos; Diosque, Patricio; Triana Chavez, Omar; Aznar, Christine; Russomando, Graciela; Büscher, Philippe; Assal, Azzedine; Guhl, Felipe; Sosa Estani, Sergio; DaSilva, Alexandre; Britto, Constança; Luquetti, Alejandro; Ladzins, Janis

2011-01-01

Background A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. Methodology/Findings An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10−3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories. Conclusion/Significance This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples. PMID:21264349

Analysis of multiple enteric viral targets as sewage markers in coral reefs

USGS Publications Warehouse

Lipp, Erin K.; Futch, J. Carrie; Griffin, Dale W.

2007-01-01

Water and coral mucus samples were collected from throughout the Florida Keys National Marine Sanctuary and the Dry Tortugas for three years and were analyzed for human enteric viruses (enteroviruses, noroviruses, hepatitis A virus and adenoviruses) as conservative markers of human sewage using molecular methods. Of the 100 coral and water samples collected, 40 contained genetic material from one or more human enteric viruses. DNA-based adenoviruses were detected widely, in 37.8% of samples and at 91% of stations, including ‘pristine’ reefs in the Dry Tortugas; however, the detection rate was ⩽12% for the RNA-based enteroviruses and noroviruses (hepatitis A virus was never detected). The disparity between the prevalence of RNA- and DNA-based viruses suggests the need for additional work to determine the utility of adenovirus as marker of human sewage.

[On the use of FTA technology for collection, archieving, and molecular analysis of microsporidia dna from clinical stool samples].

PubMed

Sokolova, O I; Dem'ianov, A V; Bovers, L S; Did'e, E S; Sokolova, Iu Ia

2011-01-01

The FTA technology was applied for sampling, archiving, and molecular analysis of the DNA isolated from stool samples to diagnose and identify microsporidia, the intracellular opportunistic parasites which induce malabsortion syndrome in immunosuppressed humans, particularly in patients with AIDS. Microsporidia DNA was successfully amplified in 6 of 50 stool samples of HIV-positive patients of the S. P. Botkin Memorial Infectious Disease Hospital (St. Petersburg) applied to FTA cards (FTA-Cars, Whatman Inc. Florham Park, NJ, USA). Amplicons (the fragments of rDNA) were directly sequenced, and microsporidia species--Encephalitozoon intestinalis, E. cuniculi, E. hellem, and Enterocytozoon bieneusi--were identified in Genbank by NCBI BLAST program. The FTA method of DNA immobilization is especially promising for epidemiological and field population studies which involve genotyping of microsporidia species and isolates.

A sensitive method to quantify human cell-free circulating DNA in blood: relevance to myocardial infarction screening.

PubMed

Jing, Rong-Rong; Wang, Hui-Min; Cui, Ming; Fang, Meng-Kang; Qiu, Xiao-Jun; Wu, Xin-Hua; Qi, Jin; Wang, Yue-Guo; Zhang, Lu-Rong; Zhu, Jian-Hua; Ju, Shao-Qing

2011-09-01

Human cell-free circulating DNA (cf-DNA) derived mainly from cell apoptosis and necrosis can be measured by a variety of laboratory techniques, but almost all of these methods require sample preparation. We have developed a branched DNA (bDNA)-based Alu assay for quantifying cf-DNA in myocardial infarction (MI) patients. A total of 82 individuals were included in the study; 22 MI and 60 normal controls. cf-DNA was quantified using a bDNA-based Alu assay. cf-DNA was higher in serum compared to plasma and there was a difference between genders. cf-DNA was significantly higher in MI patients compared to the controls. There was no correlation between cf-DNA and creatine kinase-MB (CK-MB), troponin I (cTnI) or myoglobin (MYO). In serial specimens, cf-DNA was sensitive and peaked earlier than cTnI. The bDNA-based Alu assay is a novel method for quantifying human cf-DNA. Increased cf-DNA in MI patients might complement cTnI, CK-MB and MYO in a multiple marker format. Copyright © 2011 The Canadian Society of Clinical Chemists. All rights reserved.

Molecular and serological study of rickettsial infection in humans, and in wild and farm animals, in the province of Burgos, Spain.

PubMed

Lledó, Lourdes; Domínguez-Peñafiel, Gerardo; Giménez-Pardo, Consuelo; Gegúndez, Isabel; González, Rosario; Saz, José Vicente

2014-06-01

Limited information is available on the presence of rickettsial infection in humans and animal reservoirs in Spain. Exposure to spotted fever group rickettsia in healthy humans and in farm and wild animals in the Province of Burgos, Spain, was examined by serological methods. Rickettsial DNA was also sought by PCR in animal samples. Of 102 human serum samples examined by indirect immunofluorescence assays (IFA), 5.88% were positive for antibodies against Rickettsia conorii (titers 1/128-1/512). Significant differences were detected in human seroprevalence with respect to age. In further IFAs, 102 out of 375 (27.2%) serum samples from the wild animals reacted with R. conorii antigens (titers 1/64-1/1024); 32 out of 281 (11.38%) samples from farm animals were also positive for R. conorii (titers 1/64-1/2048). The prevalence detected among total wild animals was significantly higher than among total farm animals. No rickettsial DNA was found by PCR in any farm or wild animal sample.

Development of Cross-Assembly Phage PCR-Based Methods ...

EPA Pesticide Factsheets

Technologies that can characterize human fecal pollution in environmental waters offer many advantages over traditional general indicator approaches. However, many human-associated methods cross-react with non-human animal sources and lack suitable sensitivity for fecal source identification applications. The genome of a newly discovered bacteriophage (~97 kbp), the Cross-Assembly phage or “crAssphage”, assembled from a human gut metagenome DNA sequence library is predicted to be both highly abundant and predominately occur in human feces suggesting that this double stranded DNA virus may be an ideal human fecal pollution indicator. We report the development of two human-associated crAssphage endpoint PCR methods (crAss056 and crAss064). A shotgun strategy was employed where 384 candidate primers were designed to cover ~41 kbp of the crAssphage genome deemed favorable for method development based on a series of bioinformatics analyses. Candidate primers were subjected to three rounds of testing to evaluate assay optimization, specificity, limit of detection (LOD95), geographic variability, and performance in environmental water samples. The top two performing candidate primer sets exhibited 100% specificity (n = 70 individual samples from 8 different animal species), >90% sensitivity (n = 10 raw sewage samples from different geographic locations), LOD95 of 0.01 ng/µL of total DNA per reaction, and successfully detected human fecal pollution in impaired envi

Downregulation of external death receptor genes FAS and DR5 in colorectal cancer samples positive for human papillomavirus infection.

PubMed

Karbasi, Ashraf; Borhani, Nasim; Daliri, Karim; Kazemi, Bahram; Manoochehri, Mehdi

2015-06-01

Human papillomaviruses (HPV) have frequently been detected in colorectal cancer tumor samples, and may play a role in the pathogenesis of colorectal cancer. This study was designed to investigate the presence of DNA and RNA for the high-risk HPV genotypes 16 and 18 in samples of colorectal cancer tumors and adjacent normal tissues. We also investigated the expression of proapoptotic genes in HPV-positive colorectal tumors compared to normal tissue samples. Samples of tumoral and adjacent normal tissues were fresh-frozen, and HPV DNA was identified by nested and semiquantitative PCR. Real time PCR was used to quantitatively compare the expression of HPV-18 E6 and nine proapoptotic genes in HPV-positive tumors and samples of adjacent normal tissue. HPV-16 DNA was found in 10.5% of the tumor samples, and HPV-18 DNA was found in 23.6% of the samples. Real time PCR results showed lower expression of the E6 gene in HPV-positive tumors than in adjacent normal tissue. The expression of two proapoptotic genes, FAS and DR5, was significantly lower in tumor samples than in adjacent normal tissues. HPV infection, especially HPV-18, may play a role in colorectal cancer tumorigenesis by downregulating death receptor genes and interfering with the extrinsic pathway of apoptosis. Copyright © 2015 Elsevier GmbH. All rights reserved.

Periodontal bacteria in human carotid atherothrombosis as a potential trigger for neutrophil activation.

PubMed

Rangé, Hélène; Labreuche, Julien; Louedec, Liliane; Rondeau, Philippe; Planesse, Cynthia; Sebbag, Uriel; Bourdon, Emmanuel; Michel, Jean-Baptiste; Bouchard, Philippe; Meilhac, Olivier

2014-10-01

Epidemiological, biological and clinical links between periodontal and cardiovascular diseases are now well established. Several human studies have detected bacterial DNA corresponding to periodontal pathogens in cardiovascular samples. Intraplaque hemorrhage has been associated with a higher risk of atherosclerotic plaque rupture, potentially mediated by neutrophil activation. In this study, we hypothesized that plaque composition may be related to periodontal pathogens. Carotid culprit plaque samples were collected from 157 patients. Macroscopic characterization was performed at the time of collection: presence of blood, lipid core, calcification and fibrosis. Markers of neutrophil activation released by carotid samples were quantified (myeloperoxidase or MPO, cell-free DNA and DNA-MPO complexes). PCR analysis using specific primers for Porphyromonas gingivalis, Aggregatibacter actinomycetemcommitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia was used to detect DNA from periodontal pathogens in carotid tissues. In addition, bacterial lipopolysaccharide (LPS) and Immunoglobulins G against T. forsythia were quantified in atherosclerotic carotid conditioned medium. Intraplaque hemorrhage was present in 73/157 carotid samples and was associated with neutrophil activation, reflected by the release of MPO, cell-free DNA and MPO-DNA complexes. LPS levels were also linked to intraplaque hemorrhage but not with the neutrophil activation markers. Seventy-three percent of the carotid samples were positive for periodontal bacterial DNA. Furthermore, hemoglobin levels were associated with the detection of T. forsythia and neutrophil activation/inflammation markers. This study suggests a potential role of periodontal microorganisms, especially T. forsythia, in neutrophil activation within hemorrhagic atherosclerotic carotid plaques. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

High-Throughput DNA sequencing of ancient wood.

PubMed

Wagner, Stefanie; Lagane, Frédéric; Seguin-Orlando, Andaine; Schubert, Mikkel; Leroy, Thibault; Guichoux, Erwan; Chancerel, Emilie; Bech-Hebelstrup, Inger; Bernard, Vincent; Billard, Cyrille; Billaud, Yves; Bolliger, Matthias; Croutsch, Christophe; Čufar, Katarina; Eynaud, Frédérique; Heussner, Karl Uwe; Köninger, Joachim; Langenegger, Fabien; Leroy, Frédéric; Lima, Christine; Martinelli, Nicoletta; Momber, Garry; Billamboz, André; Nelle, Oliver; Palomo, Antoni; Piqué, Raquel; Ramstein, Marianne; Schweichel, Roswitha; Stäuble, Harald; Tegel, Willy; Terradas, Xavier; Verdin, Florence; Plomion, Christophe; Kremer, Antoine; Orlando, Ludovic

2018-03-01

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high-throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long-term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human-induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro-evolutionary response of trees to climate change and human forest management. © 2018 John Wiley & Sons Ltd.

A case study characterizing animal fecal sources in surface water using a mitochondrial DNA marker.

PubMed

Bucci, John P; Shattuck, Michelle D; Aytur, Semra A; Carey, Richard; McDowell, William H

2017-08-01

Water quality impairment by fecal waste in coastal watersheds is a public health issue. The present study provided evidence for the use of a mitochondrial (mtDNA) marker to detect animal fecal sources in surface water. The accurate identification of fecal pollution is based on the notion that fecal microorganisms preferentially inhabit a host animal's gut environment. In contrast, mtDNA host-specific markers are inherent to eukaryotic host cells, which offers the advantage by detecting DNA from the host rather than its fecal bacteria. The present study focused on sampling water presumably from non-point sources (NPS), which can increase bacterial and nitrogen concentrations to receiving water bodies. Stream sampling sites located within the Piscataqua River Watershed (PRW), New Hampshire, USA, were sampled from a range of sites that experienced nitrogen inputs such as sewer and septic systems and suburban runoff. Three mitochondrial (mtDNA) gene marker assays (human, bovine, and canine) were tested from surface water. Nineteen sites were sampled during an 18-month period. Analyses of the combined single and multiplex assay results showed that the proportion of occurrence was highest for bovine (15.6%; n = 77) compared to canine (5.6%; n = 70) and human (5.7%; n = 107) mtDNA gene markers. For the human mtDNA marker, there was a statistically significant relationship between presence vs. absence and land use (Fisher's test p = 0.0031). This result was evident particularly for rural suburban septic, which showed the highest proportion of presence (19.2%) compared to the urban sewered (3.3%), suburban sewered (0%), and agricultural (0%) as well as forested septic (0%) sites. Although further testing across varied land use is needed, our study provides evidence for using the mtDNA marker in large watersheds.

Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML.

PubMed

Liyanage, Sanduni U; Hurren, Rose; Voisin, Veronique; Bridon, Gaëlle; Wang, Xiaoming; Xu, ChangJiang; MacLean, Neil; Siriwardena, Thirushi P; Gronda, Marcela; Yehudai, Dana; Sriskanthadevan, Shrivani; Avizonis, Daina; Shamas-Din, Aisha; Minden, Mark D; Bader, Gary D; Laposa, Rebecca; Schimmer, Aaron D

2017-05-11

Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2'3'-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML. © 2017 by The American Society of Hematology.

Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML

PubMed Central

Liyanage, Sanduni U.; Hurren, Rose; Voisin, Veronique; Bridon, Gaëlle; Wang, Xiaoming; Xu, ChangJiang; MacLean, Neil; Siriwardena, Thirushi P.; Gronda, Marcela; Yehudai, Dana; Sriskanthadevan, Shrivani; Avizonis, Daina; Shamas-Din, Aisha; Minden, Mark D.; Bader, Gary D.; Laposa, Rebecca

2017-01-01

Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2′3′-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2′3′-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML. PMID:28283480

Diagnostic value of amplification of human cytomegalovirus DNA from gastrointestinal biopsies from human immunodeficiency virus-infected patients.

PubMed

Cotte, L; Drouet, E; Bissuel, F; Denoyel, G A; Trepo, C

1993-08-01

In order to assess the value of human cytomegalovirus (HCMV) DNA amplification of gastrointestinal biopsies, we studied 57 human immunodeficiency virus-infected patients with and without gastrointestinal HCMV diseases. After DNA extraction, a 406-bp fragment from the unique short region of the HCMV genome was amplified by 35 cycles of polymerase chain reaction (PCR) and semiquantified from 80 to 80,000 HCMV genomic copies. Among 12 non-AIDS patients, the PCR assay was negative for 11 of 12 duodenal and 8 of 8 colorectal samples. It was also negative for 28 of 31 duodenal and 12 of 15 colorectal samples from 31 AIDS patients without gastrointestinal HCMV diseases. Among 14 AIDS patients with gastrointestinal HCMV diseases, the PCR assay was positive for 12 of 12 patients with HCMV duodenitis and for 13 of 13 patients with HCMV colitis. Results were dichotomized between high and low HCMV-DNA copy numbers. For duodenitis, sensitivity was 92% and specificity was 100%. For colitis, sensitivity was 92% and specificity was 93%. Specificity and sensitivity were not influenced by shedding status for HCMV or by other gastrointestinal infections. HCMV DNA amplification of gastrointestinal biopsies is a sensitive and specific tool for the diagnosis of gastrointestinal HCMV diseases in AIDS patients.

Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal

PubMed Central

Skoglund, Pontus; Northoff, Bernd H.; Shunkov, Michael V.; Derevianko, Anatoli P.; Pääbo, Svante; Krause, Johannes; Jakobsson, Mattias

2014-01-01

One of the main impediments for obtaining DNA sequences from ancient human skeletons is the presence of contaminating modern human DNA molecules in many fossil samples and laboratory reagents. However, DNA fragments isolated from ancient specimens show a characteristic DNA damage pattern caused by miscoding lesions that differs from present day DNA sequences. Here, we develop a framework for evaluating the likelihood of a sequence originating from a model with postmortem degradation—summarized in a postmortem degradation score—which allows the identification of DNA fragments that are unlikely to originate from present day sources. We apply this approach to a contaminated Neandertal specimen from Okladnikov Cave in Siberia to isolate its endogenous DNA from modern human contaminants and show that the reconstructed mitochondrial genome sequence is more closely related to the variation of Western Neandertals than what was discernible from previous analyses. Our method opens up the potential for genomic analysis of contaminated fossil material. PMID:24469802

MethHC: a database of DNA methylation and gene expression in human cancer.

PubMed

Huang, Wei-Yun; Hsu, Sheng-Da; Huang, Hsi-Yuan; Sun, Yi-Ming; Chou, Chih-Hung; Weng, Shun-Long; Huang, Hsien-Da

2015-01-01

We present MethHC (http://MethHC.mbc.nctu.edu.tw), a database comprising a systematic integration of a large collection of DNA methylation data and mRNA/microRNA expression profiles in human cancer. DNA methylation is an important epigenetic regulator of gene transcription, and genes with high levels of DNA methylation in their promoter regions are transcriptionally silent. Increasing numbers of DNA methylation and mRNA/microRNA expression profiles are being published in different public repositories. These data can help researchers to identify epigenetic patterns that are important for carcinogenesis. MethHC integrates data such as DNA methylation, mRNA expression, DNA methylation of microRNA gene and microRNA expression to identify correlations between DNA methylation and mRNA/microRNA expression from TCGA (The Cancer Genome Atlas), which includes 18 human cancers in more than 6000 samples, 6548 microarrays and 12 567 RNA sequencing data. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genetic polymorphisms in prehistoric Pacific islanders determined by analysis of ancient bone DNA.

PubMed

Hagelberg, E; Clegg, J B

1993-05-22

A previously characterized Asian-specific mitochondrial DNA (mtDNA) length mutation has been detected in DNA isolated from prehistoric human bones from Polynesia, including Hawaii, Chatham Islands and Society Islands. In contrast, the Asian mutation was absent in skeletal samples from the Melanesian archipelagos of New Britain and Vanuatu and in the oldest samples from Fiji, Tonga and Samoa in the central Pacific (2700-1600 years BP) although it was present in a more recent prehistoric sample from Tonga. These results, augmented by informative DNA sequence data from the hypervariable region of mtDNA, fail to support current views that the central Pacific was settled directly by voyagers from island Southeast Asia, the putative ancestors of modern Polynesians. An earlier occupation by peoples from the neighbouring Melanesian archipelagos seems more likely.

[Whole Genome Sequencing of Human mtDNA Based on Ion Torrent PGM™ Platform].

PubMed

Cao, Y; Zou, K N; Huang, J P; Ma, K; Ping, Y

2017-08-01

To analyze and detect the whole genome sequence of human mitochondrial DNA （mtDNA） by Ion Torrent PGM™ platform and to study the differences of mtDNA sequence in different tissues. Samples were collected from 6 unrelated individuals by forensic postmortem examination, including chest blood, hair, costicartilage, nail, skeletal muscle and oral epithelium. Amplification of whole genome sequence of mtDNA was performed by 4 pairs of primer. Libraries were constructed with Ion Shear™ Plus Reagents kit and Ion Plus Fragment Library kit. Whole genome sequencing of mtDNA was performed using Ion Torrent PGM™ platform. Sanger sequencing was used to determine the heteroplasmy positions and the mutation positions on HVⅠ region. The whole genome sequence of mtDNA from all samples were amplified successfully. Six unrelated individuals belonged to 6 different haplotypes. Different tissues in one individual had heteroplasmy difference. The heteroplasmy positions and the mutation positions on HVⅠ region were verified by Sanger sequencing. After a consistency check by the Kappa method, it was found that the results of mtDNA sequence had a high consistency in different tissues. The testing method used in present study for sequencing the whole genome sequence of human mtDNA can detect the heteroplasmy difference in different tissues, which have good consistency. The results provide guidance for the further applications of mtDNA in forensic science. Copyright© by the Editorial Department of Journal of Forensic Medicine

Tears from children with chronic hepatitis B virus (HBV) infection are infectious vehicles of HBV transmission: experimental transmission of HBV by tears, using mice with chimeric human livers.

PubMed

Komatsu, Haruki; Inui, Ayano; Sogo, Tsuyoshi; Tateno, Akihiko; Shimokawa, Reiko; Fujisawa, Tomoo

2012-08-15

Body fluids such as saliva, urine, sweat, and tears from hepatitis B virus (HBV) carriers are potential sources of HBV transmission. Thirty-nine children and 8 adults who were chronically infected with HBV were enrolled. Real-time polymerase chain reaction was used for the quantification of HBV DNA. HBV DNA was detected in 73.7% of urine samples (14 of 19), 86.8% of saliva samples (33 of 38), 100% of tear samples (11 of 11), and 100% of sweat samples (9 of 9). Mean HBV DNA levels (±SD) in urine, saliva, tears, and sweat were 4.3 ± 1.1 log copies/mL, 5.9 ± 1.2 log copies/mL, 6.2 ± 0.7 log copies/mL, and 5.2 ± 0.6 log copies/mL, respectively. A statistically significant correlation was observed between the HBV DNA level in serum specimens and HBV DNA levels in saliva and tear specimens (r = 0.88; P < .001). Tear specimens from a child were injected intravenously into 2 human hepatocyte-transplanted chimeric mice. One week after inoculation, both chimeric mice had serum positive for HBV DNA. The levels of HBV DNA in tear specimens from young children were high. Tears were confirmed to be infectious, using chimeric mice. Strict precautions should be taken against direct contact with body fluids from HBV carriers with high-level viremia.

DNA and RNA profiling of excavated human remains with varying postmortem intervals.

PubMed

van den Berge, M; Wiskerke, D; Gerretsen, R R R; Tabak, J; Sijen, T

2016-11-01

When postmortem intervals (PMIs) increase such as with longer burial times, human remains suffer increasingly from the taphonomic effects of decomposition processes such as autolysis and putrefaction. In this study, various DNA analysis techniques and a messenger RNA (mRNA) profiling method were applied to examine for trends in nucleic acid degradation and the postmortem interval. The DNA analysis techniques include highly sensitive DNA quantitation (with and without degradation index), standard and low template STR profiling, insertion and null alleles (INNUL) of retrotransposable elements typing and mitochondrial DNA profiling. The used mRNA profiling system targets genes with tissue specific expression for seven human organs as reported by Lindenbergh et al. (Int J Legal Med 127:891-900, 27) and has been applied to forensic evidentiary traces but not to excavated tissues. The techniques were applied to a total of 81 brain, lung, liver, skeletal muscle, heart, kidney and skin samples obtained from 19 excavated graves with burial times ranging from 4 to 42 years. Results show that brain and heart are the organs in which both DNA and RNA remain remarkably stable, notwithstanding long PMIs. The other organ tissues either show poor overall profiling results or vary for DNA and RNA profiling success, with sometimes DNA and other times RNA profiling being more successful. No straightforward relations were observed between nucleic acid profiling results and the PMI. This study shows that not only DNA but also RNA molecules can be remarkably stable and used for profiling of long-buried human remains, which corroborate forensic applications. The insight that the brain and heart tissues tend to provide the best profiling results may change sampling policies in identification cases of degrading cadavers.

Forensic DNA typing from teeth using demineralized root tips.

PubMed

Corrêa, Heitor Simões Dutra; Pedro, Fabio Luis Miranda; Volpato, Luiz Evaristo Ricci; Pereira, Thiago Machado; Siebert Filho, Gilberto; Borges, Álvaro Henrique

2017-11-01

Teeth are widely used samples in forensic human genetic identification due to their persistence and practical sampling and processing. Their processing, however, has changed very little in the last 20 years, usually including powdering or pulverization of the tooth. The objective of this study was to present demineralized root tips as DNA sources while, at the same time, not involving powdering the samples or expensive equipment for teeth processing. One to five teeth from each of 20 unidentified human bodies recovered from midwest Brazil were analyzed. Whole teeth were demineralized in EDTA solution with daily solution change. After a maximum of approximately seven days, the final millimeters of the root tip was excised. This portion of the sample was used for DNA extraction through a conventional organic protocol. DNA quantification and STR amplification were performed using commercial kits followed by capillary electrophoresis on 3130 or 3500 genetic analyzers. For 60% of the unidentified bodies (12 of 20), a full genetic profile was obtained from the extraction of the first root tip. By the end of the analyses, full genetic profiles were obtained for 85% of the individuals studied, of which 80% were positively identified. This alternative low-tech approach for postmortem teeth processing is capable of extracting DNA in sufficient quantity and quality for forensic casework, showing that root tips are viable nuclear DNA sources even after demineralization. Copyright © 2017 Elsevier B.V. All rights reserved.

DNA methylation signature of human fetal alcohol spectrum disorder.

PubMed

Portales-Casamar, Elodie; Lussier, Alexandre A; Jones, Meaghan J; MacIsaac, Julia L; Edgar, Rachel D; Mah, Sarah M; Barhdadi, Amina; Provost, Sylvie; Lemieux-Perreault, Louis-Philippe; Cynader, Max S; Chudley, Albert E; Dubé, Marie-Pierre; Reynolds, James N; Pavlidis, Paul; Kobor, Michael S

2016-01-01

Prenatal alcohol exposure is the leading preventable cause of behavioral and cognitive deficits, which may affect between 2 and 5 % of children in North America. While the underlying mechanisms of alcohol's effects on development remain relatively unknown, emerging evidence implicates epigenetic mechanisms in mediating the range of symptoms observed in children with fetal alcohol spectrum disorder (FASD). Thus, we investigated the effects of prenatal alcohol exposure on genome-wide DNA methylation in the NeuroDevNet FASD cohort, the largest cohort of human FASD samples to date. Genome-wide DNA methylation patterns of buccal epithelial cells (BECs) were analyzed using the Illumina HumanMethylation450 array in a Canadian cohort of 206 children (110 FASD and 96 controls). Genotyping was performed in parallel using the Infinium HumanOmni2.5-Quad v1.0 BeadChip. After correcting for the effects of genetic background, we found 658 significantly differentially methylated sites between FASD cases and controls, with 41 displaying differences in percent methylation change >5 %. Furthermore, 101 differentially methylated regions containing two or more CpGs were also identified, overlapping with 95 different genes. The majority of differentially methylated genes were highly expressed at the level of mRNA in brain samples from the Allen Brain Atlas, and independent DNA methylation data from cortical brain samples showed high correlations with BEC DNA methylation patterns. Finally, overrepresentation analysis of genes with up-methylated CpGs revealed a significant enrichment for neurodevelopmental processes and diseases, such as anxiety, epilepsy, and autism spectrum disorders. These findings suggested that prenatal alcohol exposure is associated with distinct DNA methylation patterns in children and adolescents, raising the possibility of an epigenetic biomarker of FASD.

Determination of the Biological Form of Human Cytomegalovirus DNA in the Plasma of Solid-Organ Transplant Recipients.

PubMed

Tong, Yupin; Pang, Xiaoli L; Mabilangan, Curtis; Preiksaitis, Jutta K

2017-04-01

Whether cytomegalovirus (CMV) DNA exists in plasma as virion-associated or free DNA is uncertain. An assay combining DNase I digestion and CMV quantitative polymerase chain reaction (DNase-CMV-qPCR) was developed to differentiate free naked DNA from virion DNA. One hundred three frozen and 10 fresh CMV DNA-positive plasma samples from solid-organ transplant recipients (SOTRs) were tested. Three sets of paired qPCR (P-qPCR) assays with amplicons of variable length were used to study CMV DNA fragmentation in 20 SOTR plasma samples, viral stocks (Towne, Merlin, AD169) and the first World Health Organization (WHO) international standard (IS) for CMV DNA. In all plasma samples, 98.8%-100% of CMV DNA was free DNA; this was the only form in 93 of 103 (90.3%) frozen and all 10 fresh samples tested using DNase-CMV-qPCR. Low levels of virion CMV DNA were found in 10 of 103 (9.7%) samples with higher total DNA load. Cytomegalovirus DNA results were highly reproducible for 3 CMV virus stocks and WHO IS (P > .80), tested by three sets of paired q-PCR. However, for the 20 SOTR plasma samples, the smaller amplicon assay result was 2.6-fold, 3.4-fold, and 6.5-fold higher than the longer amplicion result (P < .001). Cytomegalovirus DNA in SOTR plasma is almost exclusively free DNA, highly fragmented, and not virion associated. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Use of FTA elute card impregnated with cervicovaginal sample directly into the amplification reaction increases the detection of human papillomavirus DNA.

PubMed

Santos, Carla R; Franciscatto, Laura G; Barcellos, Regina B; Almeida, Sabrina E M; Rossetti, Maria Lucia R

2012-01-01

This study aimed to evaluate the use of the FTA elute card(TM) impregnated with cervicovaginal sample directly in the PCR amplification for detection of HPV-DNA. The results were compared to a reference technique. This method was more efficient than the protocol indicated by the manufacturer, identifying 91.7% against 54.2% of the positive samples.

Autoclave sterilization of instruments used on women with cervical neoplasia is an effective method of eradicating residual human papillomavirus DNA: a polymerase chain reaction-based evaluation.

PubMed

Estes, Jacob M; Kirby, Tyler O; Huh, Warner K

2007-01-01

To determine whether autoclave sterilization eradicates human papillomavirus (HPV) DNA on specula and instruments used to treat women with cervical neoplasia. Specula and instruments used in two referral colposcopy clinics were evaluated to determine the PGMY9/11 primer system's ability to amplify residual HPV DNA. Each speculum and instrument was sampled with a Dacron swab and stored in PreservCyt solution (Cytyc Corporation, Marlborough, MA) at 4 degrees C. DNA amplification was performed under standard conditions with appropriate controls followed by HPV typing using the reverse line blot test (Roche Molecular Systems, Alameda, CA). Once validated, the same polymerase chain reaction method was used on autoclave-sterilized specula and biopsy instruments and heated glass bead- and Cidex bath (Johnson & Johnson, New Brunswick, NJ)-sterilized instruments. All results, with appropriate positive and negative controls, were confirmed in triplicate. A total of 140 instruments (70 used and 70 autoclaved) were sampled for residual HPV DNA. Five samples in the contaminated specula arm were excluded from analysis secondary to insufficient sampling. Of the remaining samples, 52.3% (34/65) of contaminated instruments-both specula and biopsy instruments-had detectable HPV DNA. Fifty-five percent of contaminated biopsy instruments (11/20) were positive and 51.1% of contaminated specula (23/45) were positive. All 70 autoclaved samples (50 specula and 20 biopsy instruments) were negative for residual HPV DNA or beta-globin. One instrument in the glass bead and Cidex group that was presumed sterile was positive for HPV 16 DNA. The PGMY9/11 primer system is an effective method to detect residual HPV DNA. Autoclave sterilization appears to eradicate HPV DNA to levels undetectable with this sensitive assay, whereas heated glass beads followed by Cidex bath appears to be inadequate methods. These results suggest that autoclave sterilization is effective when using nondisposable instruments and should be the method of choice in studies using polymerase chain reaction-based amplification of HPV DNA.

Evaluation and Validation of a Real-Time PCR Assay for Detection and Quantitation of Human Adenovirus 14 from Clinical Samples

DTIC Science & Technology

2009-09-01

Legionella Pneumophila (ATCC 33152) were acquired from the American Type Culture Collection (ATCC; Manassas, VA, USA). Conventional PCR testing The primers... Legionella Pneumophila – One nanogram of genomic DNA was used as a DNA template in each assay. Assays were performed in triplicate. Samples were negative at

Metagenomic Analysis of Milk of Healthy and Mastitis-Suffering Women.

PubMed

Jiménez, Esther; de Andrés, Javier; Manrique, Marina; Pareja-Tobes, Pablo; Tobes, Raquel; Martínez-Blanch, Juan F; Codoñer, Francisco M; Ramón, Daniel; Fernández, Leónides; Rodríguez, Juan M

2015-08-01

Some studies have been conducted to assess the composition of the bacterial communities inhabiting human milk, but they did not evaluate the presence of other microorganisms, such as fungi, archaea, protozoa, or viruses. This study aimed to compare the metagenome of human milk samples provided by healthy and mastitis-suffering women. DNA was isolated from human milk samples collected from 10 healthy women and 10 women with symptoms of lactational mastitis. Shotgun libraries from total extracted DNA were constructed and the libraries were sequenced by 454 pyrosequencing. The amount of human DNA sequences was ≥ 90% in all the samples. Among the bacterial sequences, the predominant phyla were Proteobacteria, Firmicutes, and Bacteroidetes. The healthy core microbiome included the genera Staphylococcus, Streptococcus, Bacteroides, Faecalibacterium, Ruminococcus, Lactobacillus, and Propionibacterium. At the species level, a high degree of inter-individual variability was observed among healthy women. In contrast, Staphylococcus aureus clearly dominated the microbiome in the samples from the women with acute mastitis whereas high increases in Staphylococcus epidermidis-related reads were observed in the milk of those suffering from subacute mastitis. Fungal and protozoa-related reads were identified in most of the samples, whereas Archaea reads were absent in samples from women with mastitis. Some viral-related sequence reads were also detected. Human milk contains a complex microbial metagenome constituted by the genomes of bacteria, archaea, viruses, fungi, and protozoa. In mastitis cases, the milk microbiome reflects a loss of bacterial diversity and a high increase of the sequences related to the presumptive etiological agents. © The Author(s) 2015.

Epigenetic Pattern on the Human Y Chromosome Is Evolutionarily Conserved

PubMed Central

Meng, Hao; Agbagwa, Ikechukwu O.; Wang, Ling-Xiang; Wang, Yingzhi; Yan, Shi; Ren, Shancheng; Sun, Yinghao; Pei, Gang; Liu, Xin; Liu, Jiang; Jin, Li; Li, Hui; Sun, Yingli

2016-01-01

DNA methylation plays an important role for mammalian development. However, it is unclear whether the DNA methylation pattern is evolutionarily conserved. The Y chromosome serves as a powerful tool for the study of human evolution because it is transferred between males. In this study, based on deep-rooted pedigrees and the latest Y chromosome phylogenetic tree, we performed epigenetic pattern analysis of the Y chromosome from 72 donors. By comparing their respective DNA methylation level, we found that the DNA methylation pattern on the Y chromosome was stable among family members and haplogroups. Interestingly, two haplogroup-specific methylation sites were found, which were both genotype-dependent. Moreover, the African and Asian samples also had similar DNA methylation pattern with a remote divergence time. Our findings indicated that the DNA methylation pattern on the Y chromosome was conservative during human male history. PMID:26760298

Human papillomavirus type 2 associated with pyogenic granuloma in patients without clinical evidence of warts.

PubMed

Vázquez-Martínez, Osvaldo T; González-Betancourt, Anajulia; Barboza-Cerda, María Carmen; González-González, Sergio E; Lugo-Trampe, Ángel; Welsh, Oliverio; Rojas-Martínez, Augusto; Martínez-Rodríguez, Herminia G; Ocampo-Candiani, Jorge; Ortiz-López, Rocío

2016-07-01

Pyogenic granuloma is a non-neoplastic lesion that frequently occurs in the skin and mucous membranes of children and pregnant women. The anatomical sites of pyogenic granulomas overlap with those of wart infections caused by the human papillomavirus (HPV). This study assessed the presence of HPV DNA in pyogenic granuloma samples by polymerase chain reaction. Eighteen pyogenic granuloma biopsies from patients without a clinical history or evidence of verruca in the studied area were tested for the presence of the HPV genome. The presence of HPV DNA was screened by three independent polymerase chain reaction reactions using standard consensus primer sets targeted to the L1 or E1 consensus regions of HPV genome. The HPV DNA-positive samples were genotyped using methodologies enabling the identification of up to 30 HPVs, including oncogenic, nononcogenic, and cutaneous viral types. The HPV DNA was detected in 44.4% (eight of 18) of the samples, with HPV-2 being the only type in the eight HPV DNA-positive samples. Contamination with HPV-2 sequences throughout the entire process was reliably eliminated. This report is the first to suggest an association between HPV-2 and pyogenic granuloma. This relationship is similar to that observed between HPV-2 and nongenital warts. © 2015 The International Society of Dermatology.

More comprehensive forensic genetic marker analyses for accurate human remains identification using massively parallel DNA sequencing.

PubMed

Ambers, Angie D; Churchill, Jennifer D; King, Jonathan L; Stoljarova, Monika; Gill-King, Harrell; Assidi, Mourad; Abu-Elmagd, Muhammad; Buhmeida, Abdelbaset; Al-Qahtani, Mohammed; Budowle, Bruce

2016-10-17

Although the primary objective of forensic DNA analyses of unidentified human remains is positive identification, cases involving historical or archaeological skeletal remains often lack reference samples for comparison. Massively parallel sequencing (MPS) offers an opportunity to provide biometric data in such cases, and these cases provide valuable data on the feasibility of applying MPS for characterization of modern forensic casework samples. In this study, MPS was used to characterize 140-year-old human skeletal remains discovered at a historical site in Deadwood, South Dakota, United States. The remains were in an unmarked grave and there were no records or other metadata available regarding the identity of the individual. Due to the high throughput of MPS, a variety of biometric markers could be typed using a single sample. Using MPS and suitable forensic genetic markers, more relevant information could be obtained from a limited quantity and quality sample. Results were obtained for 25/26 Y-STRs, 34/34 Y SNPs, 166/166 ancestry-informative SNPs, 24/24 phenotype-informative SNPs, 102/102 human identity SNPs, 27/29 autosomal STRs (plus amelogenin), and 4/8 X-STRs (as well as ten regions of mtDNA). The Y-chromosome (Y-STR, Y-SNP) and mtDNA profiles of the unidentified skeletal remains are consistent with the R1b and H1 haplogroups, respectively. Both of these haplogroups are the most common haplogroups in Western Europe. Ancestry-informative SNP analysis also supported European ancestry. The genetic results are consistent with anthropological findings that the remains belong to a male of European ancestry (Caucasian). Phenotype-informative SNP data provided strong support that the individual had light red hair and brown eyes. This study is among the first to genetically characterize historical human remains with forensic genetic marker kits specifically designed for MPS. The outcome demonstrates that substantially more genetic information can be obtained from the same initial quantities of DNA as that of current CE-based analyses.

Strategy for Sensitive and Specific Detection of Yersinia pestis in Skeletons of the Black Death Pandemic

PubMed Central

Seifert, Lisa; Harbeck, Michaela; Thomas, Astrid; Hoke, Nadja; Zöller, Lothar; Wiechmann, Ingrid; Grupe, Gisela; Scholz, Holger C.; Riehm, Julia M.

2013-01-01

Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14th century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study. PMID:24069445

Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.

PubMed

Seifert, Lisa; Harbeck, Michaela; Thomas, Astrid; Hoke, Nadja; Zöller, Lothar; Wiechmann, Ingrid; Grupe, Gisela; Scholz, Holger C; Riehm, Julia M

2013-01-01

Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14(th) century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study.

Comparison of commercially-available preservatives for maintaining the integrity of bacterial DNA in human milk.

PubMed

Lackey, Kimberly A; Williams, Janet E; Price, William J; Carrothers, Janae M; Brooker, Sarah L; Shafii, Bahman; McGuire, Mark A; McGuire, Michelle K

2017-10-01

Inhibiting changes to bacteria in human milk between sample collection and analysis is necessary for unbiased characterization of the milk microbiome. Although cold storage is considered optimal, alternative preservation is sometimes necessary. The objective of this study was to compare the effectiveness of several commercially-available preservatives with regard to maintaining bacterial DNA in human milk for delayed microbiome analysis. Specifically, we compared Life Technologies' RNAlater® stabilization solution, Biomatrica's DNAgard® Saliva, Advanced Instruments' Broad Spectrum Microtabs II™, and Norgen Biotek Corporation's Milk DNA Preservation and Isolation Kit. Aliquots of 8 pools of human milk were treated with each preservative. DNA was extracted immediately and at 1, 2, 4, and 6wk, during which time milk was held at 37°C. The V1-V3 region of the bacterial 16S rRNA gene was amplified and sequenced. Changes in bacterial community structure and diversity over time were evaluated. Comparable to other studies, the most abundant genera were Streptococcus (33.3%), Staphylococcus (14.0%), Dyella (6.3%), Pseudomonas (3.0%), Veillonella (2.5%), Hafnia (2.0%), Prevotella (1.7%), Rhodococcus (1.6%), and Granulicatella (1.4%). Overall, use of Norgen's Milk DNA Preservation and Isolation Kit best maintained the consistency of the bacterial community structure. Total DNA, diversity, and evenness metrics were also highest in samples preserved with this method. When collecting human milk for bacterial community analysis in field conditions where cold storage is not available, our results suggest that Norgen's Milk DNA Preservation and Isolation Kit may be a useful method, at least for a period of 2weeks. Copyright © 2017 Elsevier B.V. All rights reserved.

Clinical forensic sample collection techniques following consensual intercourse in volunteers - cervical canal brush compared to conventional swabs.

PubMed

Joki-Erkkilä, Minna; Tuomisto, Sari; Seppänen, Mervi; Huhtala, Heini; Ahola, Arja; Rainio, Juha; Karhunen, Pekka J

2014-10-01

The purpose of the research was to evaluate gynecological evidence collection techniques; the benefit of cervical canal brush sample compared to vaginal fornix and cervical swab samples and the time frame for detecting Y-chromosomal material QiAmp DNA Mini Kit(®) and Quantifiler Y Human Male DNA Quantification Kit(®) in adult volunteers following consensual intercourse. Eighty-four adult female volunteers following consensual intercourse were recruited for the study. By combining all sample collecting techniques, 81.0% of the volunteers were Y-DNA positive. Up to 60 h the conventional swab sampling techniques detected more Y-DNA positive samples when compared to the brush technique. However, after 60 h, the cervical canal brush sample technique showed its benefit by detecting 27.3% (6/22) of Y-DNA positive samples, which were Y-DNA negative in both conventional swab sampling techniques. By combining swab and brush techniques, 75% of the volunteers were still Y-DNA positive in 72-144 post-coital hours. The rate of measurable Y-DNA decreased approximately 3% per hour. Despite reported consensual intercourse, 6.8% (3/44) of volunteers were Y-DNA negative within 48 h. Y-DNA was not detected after 144 post-coital hours (6 days). In conclusion, the brush as a forensic evidence collection method may provide additional biological trace evidence from the cervical canal, although the best biological trace evidence collection can be obtained by combining all three sampling techniques. The time frame for gynecological forensic evidence sample collection should be considered to be at least a week if sexual violence is suspected. Copyright © 2014 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

Detection of mitochondrial DNA with the compact bead array sensor system (cBASS)

NASA Astrophysics Data System (ADS)

Mulvaney, Shawn P.; Ibe, Carol N.; Caldwell, Jane M.; Levine, Jay F.; Whitman, Lloyd J.; Tamanaha, Cy R.

2009-02-01

Enteric pathogens are a significant contaminant in surface waters used for recreation, fish and shellfish harvesting, crop irrigation, and human consumption. The need for water monitoring becomes more pronounced when industrial, agricultural, and residential lands are found in close proximity. Fecal contamination is particularly problematic and identification of the pollution source essential to remediation efforts. Standard monitoring for fecal contamination relies on indicator organisms, but the technique is too broad to identify the source of contamination. Instead, real-time PCR of mitochondrial DNA (mtDNA) is an emerging method for identification of the contamination source. Presented herein, we evaluate an alternative technology, the compact Bead Array Sensor System (cBASS®) and its assay approach Fluidic Force Discrimination (FFD), for the detection of mtDNA. Previously, we achieved multiplexed, attomolar detection of toxins and femtomolar detection of nucleic acids in minutes with FFD assays. More importantly, FFD assays are compatible with a variety of complex matrices and therefore potentially applicable for samples where the matrix would interfere with PCR amplification. We have designed a triplex assay for the NADH gene found in human, swine, and bovine mtDNA and demonstrated the specific detection of human mtDNA spiked into a waste water sample.

Viability and Burden of Leishmania in Extralesional Sites during Human Dermal Leishmaniasis

PubMed Central

Romero, Ibeth; Téllez, Jair; Suárez, Yazmín; Cardona, Maria; Figueroa, Roger; Zelazny, Adrian; Gore Saravia, Nancy

2010-01-01

Background The clinical and epidemiological significance of Leishmania DNA in extralesional sites is obscured by uncertainty of whether the DNA derives from viable parasites. To examine dissemination of Leishmania during active disease and the potential participation of human infection in transmission, Leishmania 7SLRNA was exploited to establish viability and estimate parasite burden in extralesional sites of dermal leishmaniasis patients. Methods The feasibility of discriminating parasite viability by PCR of Leishmania 7SLRNA was evaluated in relation with luciferase activity of luc transfected intracellular amastigotes in dose-response assays of Glucantime cytotoxicity. Monocytes, tonsil swabs, aspirates of normal skin and lesions of 28 cutaneous and 2 mucocutaneous leishmaniasis patients were screened by kDNA amplification/Southern blot. Positive samples were analyzed by quantitative PCR of Leishmania 7SLRNA genes and transcripts. Results 7SLRNA amplification coincided with luciferase activity, confirming discrimination of parasite viability. Of 22 patients presenting kDNA in extralesional samples, Leishmania 7SLRNA genes or transcripts were detected in one or more kDNA positive samples in 100% and 73% of patients, respectively. Gene and transcript copy number amplified from extralesional tissues were comparable to lesions. 7SLRNA transcripts were detected in 13/19 (68%) monocyte samples, 5/12 (42%) tonsil swabs, 4/11 (36%) normal skin aspirates, and 22/25 (88%) lesions; genes were quantifiable in 15/19 (79%) monocyte samples, 12/13 (92%) tonsil swabs, 8/11 (73%) normal skin aspirates. Conclusion Viable parasites are present in extralesional sites, including blood monocytes, tonsils and normal skin of dermal leishmaniasis patients. Leishmania 7SLRNA is an informative target for clinical and epidemiologic investigations of human leishmaniasis. PMID:20856851

Evaluation of ebselen supplementation on cryopreservation medium in human semen

PubMed Central

Khodayari Naeini, Zohreh; Hassani Bafrani, Hassan; Nikzad, Hossein

2014-01-01

Background: An effect of cryopreservation on human sperm is sublethal cryodamage, in which cell viability post-thaw is lost more rapidly at later times than in fresh cells. Objective: This study examined whether the addition of an antioxidant to cryopreservation medium could improve the post-thaw parameters and evaluation of sperm chromatin quality of cryopreserved human spermatozoa from men with normal semen parameters. Materials and Methods: Semen samples (n=35) were collected by masturbation and assessed following WHO standards. Individual samples were classified as two portions. One portion (n=10) was for elucidate the concentration of ebselen.Then the samples(n=25) were divided in to 5groups.The first aliquot remained fresh.The second aliquots was mixed with cryopreservation medium.The third aliquots were mixed with cryopreservation medium containing solvent of ebselen.The forth and fifth aliquots were mixed with cryopreservation medium containing 1.25 and 2.5 µm of ebselen.Samples were frozen and thawed samples were assessed for sperm parameters.Three-way ANOVA Multivariate measures were used to assess. According to this assesment the differences are observed in existent groups in post-thaw count, motility index, vitality staining, and morphology and DNA fragmentation. Results: After freezing the media containing of ebselen, DNA fragmentation is significantly different in comparison with control group. ebselen with 1.25 µm dose was significantly associated with post-thaw DNA fragmentation (p=0.047). Similarly ebselen with 2.5 µm dose was significantly associated with post-thaw DNA fragmentation (p=0.038). But other parameters were not altered. Conclusion: These results suggest that the addition of ebselen to cryopreservation medium doesnot improve post-thaw parameters and DNA fragmentation of sperm. PMID:24976819

Evaluation of ebselen supplementation on cryopreservation medium in human semen.

PubMed

Khodayari Naeini, Zohreh; Hassani Bafrani, Hassan; Nikzad, Hossein

2014-04-01

An effect of cryopreservation on human sperm is sublethal cryodamage, in which cell viability post-thaw is lost more rapidly at later times than in fresh cells. This study examined whether the addition of an antioxidant to cryopreservation medium could improve the post-thaw parameters and evaluation of sperm chromatin quality of cryopreserved human spermatozoa from men with normal semen parameters. Semen samples (n=35) were collected by masturbation and assessed following WHO standards. Individual samples were classified as two portions. One portion (n=10) was for elucidate the concentration of ebselen.Then the samples(n=25) were divided in to 5groups.The first aliquot remained fresh.The second aliquots was mixed with cryopreservation medium.The third aliquots were mixed with cryopreservation medium containing solvent of ebselen.The forth and fifth aliquots were mixed with cryopreservation medium containing 1.25 and 2.5 µm of ebselen.Samples were frozen and thawed samples were assessed for sperm parameters.Three-way ANOVA Multivariate measures were used to assess. According to this assesment the differences are observed in existent groups in post-thaw count, motility index, vitality staining, and morphology and DNA fragmentation. After freezing the media containing of ebselen, DNA fragmentation is significantly different in comparison with control group. ebselen with 1.25 µm dose was significantly associated with post-thaw DNA fragmentation (p=0.047). Similarly ebselen with 2.5 µm dose was significantly associated with post-thaw DNA fragmentation (p=0.038). But other parameters were not altered. These results suggest that the addition of ebselen to cryopreservation medium doesnot improve post-thaw parameters and DNA fragmentation of sperm.

Use of DNA Markers for Investigating Sources of Bacteria in Contaminated Ground Water: Wooster Township, Wayne County, Ohio

USGS Publications Warehouse

Dumouchelle, Denise H.

2006-01-01

In 2004, a public-health nuisance was declared by the Wayne County Board of Health in the Scenic Heights Drive-Batdorf Road area of Wooster Township, Wayne County, Ohio, because of concerns about the safety of water from local wells. Repeated sampling had detected the presence of fecal-indicator bacteria and elevated nitrate concentrations. In June 2006, the U.S. Geological Survey (USGS), in cooperation with the Ohio Environmental Protection Agency (Ohio EPA), collected and analyzed samples from some of the affected wells to help investigate the possibility of human-origin bacterial contamination. Water samples from 12 wells and 5 home sewage-treatment systems (HSTS) were collected. Bromide concentrations were determined in samples from the 12 wells. Samples from 5 of the 12 wells were analyzed for wastewater compounds. Total coliform, enterococci and Escherichia coli (E. coli) bacteria concentrations were determined for samples from 8 of the 12 wells. In addition, two microbial source-tracking tools that employ DNA markers were used on samples from several wells and a composite sample of water from five septic tanks. The DNA markers from the Enterococcus faecium species and the order Bacteroidales are associated with specific sources, either human or ruminant sources. Bromide concentrations ranged from 0.04 to 0.18 milligrams per liter (mg/L). No wastewater compounds were detected at concentrations above the reporting limits. Samples from the 12 wells also were collected by Ohio EPA and analyzed for chloride and nitrate. Chloride concentrations ranged from 12.6 to 61.6 mg/L and nitrate concentrations ranged from 2.34 to 11.9 mg/L (as N). Total coliforms and enterococci were detected in samples from 8 wells, at concentrations from 2 to 200 colony-forming units per 100 milliliters (CFU/100 mL) and 0.5 to 17 CFU/100 mL, respectively. E. coli were detected in samples from three of the eight wells, at concentrations of 1 or 2 CFU/100 mL. Tests for the human-specific marker of enterococci, the esp gene, were negative in the seven samples tested, including the composite sample of HSTS water. DNA with the general Bacteroidales marker was detected in samples from four wells, but the tests for both the human- and ruminant-associated markers were negative. The presence of the PCR (polymerase chain reaction) -detectable DNA for the general fecal Bacteroidales marker is indicative of fecal contamination and recently recharged water.

Smad4 loss promotes lung cancer formation but increases sensitivity to DNA topoisomerase inhibitors

PubMed Central

Kalra, Sean; Cleaver, Timothy G.; Merrick, Daniel; Wang, Xiao-Jing; Malkoski, Stephen P.

2015-01-01

Non-small cell lung cancer (NSCLC) is a common malignancy with a poor prognosis. Despite progress targeting oncogenic drivers, there are no therapies targeting tumor suppressor loss. Smad4 is an established tumor suppressor in pancreatic and colon cancer, however, the consequences of Smad4 loss in lung cancer are largely unknown. We evaluated Smad4 expression in human NSCLC samples and examined Smad4 alterations in large NSCLC datasets and found that reduced Smad4 expression is common in human NSCLC and occurs through a variety of mechanisms including mutation, homozygous deletion, and heterozygous loss. We modeled Smad4 loss in lung cancer by deleting Smad4 in airway epithelial cells and found that Smad4 deletion both initiates and promotes lung tumor development. Interestingly, both Smad4−/− mouse tumors and human NSCLC samples with reduced Smad4 expression demonstrated increased DNA damage while Smad4 knockdown in lung cancer cells reduced DNA repair and increased apoptosis after DNA damage. In addition, Smad4 deficient NSCLC cells demonstrated increased sensitivity to both chemotherapeutics that inhibit DNA topoisomerase and drugs that block double strand DNA break repair by non-homologous end joining. In sum, these studies establish Smad4 as a lung tumor suppressor and suggest that the defective DNA repair phenotype of Smad4 deficient tumors can be exploited by specific therapeutic strategies. PMID:25893305

[The validation of kit of reagents for quantitative detection of DNA of human cytomegalovirus in biological material using polymerase chain reaction technique in real time operation mode].

PubMed

Sil'veĭstrova, O Iu; Domonova, É A; Shipulina, O Iu

2014-04-01

The validation of kit of reagents destined to detection and quantitative evaluation of DNA of human cytomegalovirus in biological material using polymerase chain reaction technique in real time operation mode was implemented. The comparison was made against international WHO standard--The first WHO international standard for human cytomegalovirus to implement measures the kit of reagents "AmpliSens CMV-screen/monitor-FL" and standard sample of enterprise DNA HCMV (The central research institute of epidemiology of Rospotrebnadzor) was applied. The fivefold dilution of international WHO standard and standard sample of enterprise were carried out in concentrations of DNA HCMV from 106 to 102. The arrangement of polymerase chain reaction and analysis of results were implemented using programed amplifier with system of detection of fluorescent signal in real-time mode "Rotor-Gene Q" ("Qiagen", Germany). In the total of three series of experiments, all stages of polymerase chain reaction study included, the coefficient of translation of quantitative evaluation of DNA HCMV from copy/ml to ME/ml equal to 0.6 was introduced for this kit of reagents.

Detection of Bacterial Pathogens from Broncho-Alveolar Lavage by Next-Generation Sequencing.

PubMed

Leo, Stefano; Gaïa, Nadia; Ruppé, Etienne; Emonet, Stephane; Girard, Myriam; Lazarevic, Vladimir; Schrenzel, Jacques

2017-09-20

The applications of whole-metagenome shotgun sequencing (WMGS) in routine clinical analysis are still limited. A combination of a DNA extraction procedure, sequencing, and bioinformatics tools is essential for the removal of human DNA and for improving bacterial species identification in a timely manner. We tackled these issues with a broncho-alveolar lavage (BAL) sample from an immunocompromised patient who had developed severe chronic pneumonia. We extracted DNA from the BAL sample with protocols based either on sequential lysis of human and bacterial cells or on the mechanical disruption of all cells. Metagenomic libraries were sequenced on Illumina HiSeq platforms. Microbial community composition was determined by k-mer analysis or by mapping to taxonomic markers. Results were compared to those obtained by conventional clinical culture and molecular methods. Compared to mechanical cell disruption, a sequential lysis protocol resulted in a significantly increased proportion of bacterial DNA over human DNA and higher sequence coverage of Mycobacterium abscessus , Corynebacterium jeikeium and Rothia dentocariosa , the bacteria reported by clinical microbiology tests. In addition, we identified anaerobic bacteria not searched for by the clinical laboratory. Our results further support the implementation of WMGS in clinical routine diagnosis for bacterial identification.

Molecular evidence of simian virus 40 infections in children

NASA Technical Reports Server (NTRS)

Butel, J. S.; Arrington, A. S.; Wong, C.; Lednicky, J. A.; Finegold, M. J.

1999-01-01

Recent studies have detected simian virus 40 (SV40) DNA in certain human tumors and normal tissues. The significance of human infections by SV40, which was first discovered as a contaminant of poliovirus vaccines used between 1955 and 1963, remains unknown. The occurrence of SV40 infections in unselected hospitalized children was evaluated. Polymerase chain reaction and DNA sequence analyses were done on archival tissue specimens from patients positive for SV40 neutralizing antibody. SV40 DNA was identified in samples from 4 of 20 children (1 Wilms' tumor, 3 transplanted kidney samples). Sequence variation among SV40 regulatory regions ruled out laboratory contamination of specimens. This study shows the presence of SV40 infections in pediatric patients born after 1982.

The impact of lymphocyte isolation on induced DNA damage in human blood samples measured by the comet assay.

PubMed

Bausinger, Julia; Speit, Günter

2016-09-01

The comet assay is frequently used in human biomonitoring for the detection of exposure to genotoxic agents. Peripheral blood samples are most frequently used and tested either as whole blood or after isolation of lymphocytes (i.e. peripheral blood mononuclear cells, PBMC). To investigate a potential impact of lymphocyte isolation on induced DNA damage in human blood samples, we exposed blood ex vivo to mutagens with different modes of genotoxic action. The comet assay was performed either directly with whole blood at the end of the exposure period or with lymphocytes isolated directly after exposure. In addition to the recommended standard protocol for lymphocyte isolation, a shortened protocol was established to optimise the isolation procedure. The results indicate that the effects of induced DNA strand breaks and alkali-labile sites induced by ionising radiation and alkylants, respectively, are significantly reduced in isolated lymphocytes. In contrast, oxidative DNA base damage (induced by potassium bromate) and stable bulky adducts (induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide; BPDE) seem to be less affected. Our findings suggest that in vivo-induced DNA damage might also be reduced in isolated lymphocytes in comparison with the whole blood depending of the types of DNA damage induced. Because only small genotoxic effects can generally be expected in human biomonitoring studies with the comet assay after occupational and environmental exposure to genotoxic agents, any loss might be relevant and should be avoided. The possibility of such effects and their potential impact on variability of comet assay results in human biomonitoring should be considered when performing or evaluating such kind of studies. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Drafting human ancestry: what does the Neanderthal genome tell us about hominid evolution? Commentary on Green et al. (2010).

PubMed

Hofreiter, Michael

2011-02-01

Ten years after the first draft versions of the human genome were announced, technical progress in both DNA sequencing and ancient DNA analyses has allowed a research team around Ed Green and Svante Pääbo to complete this task from infinitely more difficult hominid samples: a few pieces of bone originating from our closest, albeit extinct, relatives, the Neanderthals. Pulling the Neanderthal sequences out of a sea of contaminating environmental DNA impregnating the bones and at the same time avoiding the problems of contamination with modern human DNA is in itself a remarkable accomplishment. However, the crucial question in the long run is, what can we learn from such genomic data about hominid evolution?

A microbial survey of the International Space Station (ISS)

PubMed Central

Lang, Jenna M.; Coil, David A.; Neches, Russell Y.; Brown, Wendy E.; Cavalier, Darlene; Severance, Mark; Hampton-Marcell, Jarrad T.; Gilbert, Jack A.

2017-01-01

Background Modern advances in sequencing technology have enabled the census of microbial members of many natural ecosystems. Recently, attention is increasingly being paid to the microbial residents of human-made, built ecosystems, both private (homes) and public (subways, office buildings, and hospitals). Here, we report results of the characterization of the microbial ecology of a singular built environment, the International Space Station (ISS). This ISS sampling involved the collection and microbial analysis (via 16S rDNA PCR) of 15 surfaces sampled by swabs onboard the ISS. This sampling was a component of Project MERCCURI (Microbial Ecology Research Combining Citizen and University Researchers on ISS). Learning more about the microbial inhabitants of the “buildings” in which we travel through space will take on increasing importance, as plans for human exploration continue, with the possibility of colonization of other planets and moons. Results Sterile swabs were used to sample 15 surfaces onboard the ISS. The sites sampled were designed to be analogous to samples collected for (1) the Wildlife of Our Homes project and (2) a study of cell phones and shoes that were concurrently being collected for another component of Project MERCCURI. Sequencing of the 16S rDNA genes amplified from DNA extracted from each swab was used to produce a census of the microbes present on each surface sampled. We compared the microbes found on the ISS swabs to those from both homes on Earth and data from the Human Microbiome Project. Conclusions While significantly different from homes on Earth and the Human Microbiome Project samples analyzed here, the microbial community composition on the ISS was more similar to home surfaces than to the human microbiome samples. The ISS surfaces are species-rich with 1,036–4,294 operational taxonomic units (OTUs per sample). There was no discernible biogeography of microbes on the 15 ISS surfaces, although this may be a reflection of the small sample size we were able to obtain. PMID:29492330

A microbial survey of the International Space Station (ISS).

PubMed

Lang, Jenna M; Coil, David A; Neches, Russell Y; Brown, Wendy E; Cavalier, Darlene; Severance, Mark; Hampton-Marcell, Jarrad T; Gilbert, Jack A; Eisen, Jonathan A

2017-01-01

Modern advances in sequencing technology have enabled the census of microbial members of many natural ecosystems. Recently, attention is increasingly being paid to the microbial residents of human-made, built ecosystems, both private (homes) and public (subways, office buildings, and hospitals). Here, we report results of the characterization of the microbial ecology of a singular built environment, the International Space Station (ISS). This ISS sampling involved the collection and microbial analysis (via 16S rDNA PCR) of 15 surfaces sampled by swabs onboard the ISS. This sampling was a component of Project MERCCURI (Microbial Ecology Research Combining Citizen and University Researchers on ISS). Learning more about the microbial inhabitants of the "buildings" in which we travel through space will take on increasing importance, as plans for human exploration continue, with the possibility of colonization of other planets and moons. Sterile swabs were used to sample 15 surfaces onboard the ISS. The sites sampled were designed to be analogous to samples collected for (1) the Wildlife of Our Homes project and (2) a study of cell phones and shoes that were concurrently being collected for another component of Project MERCCURI. Sequencing of the 16S rDNA genes amplified from DNA extracted from each swab was used to produce a census of the microbes present on each surface sampled. We compared the microbes found on the ISS swabs to those from both homes on Earth and data from the Human Microbiome Project. While significantly different from homes on Earth and the Human Microbiome Project samples analyzed here, the microbial community composition on the ISS was more similar to home surfaces than to the human microbiome samples. The ISS surfaces are species-rich with 1,036-4,294 operational taxonomic units (OTUs per sample). There was no discernible biogeography of microbes on the 15 ISS surfaces, although this may be a reflection of the small sample size we were able to obtain.

[Evaluation of 3 methods of DNA extraction from paraffin-embedded material for the amplification of genomic DNA using PCR].

PubMed

Mesquita, R A; Anzai, E K; Oliveira, R N; Nunes, F D

2001-01-01

There are several protocols reported in the literature for the extraction of genomic DNA from formalin-fixed paraffin-embedded samples. Genomic DNA is utilized in molecular analyses, including PCR. This study compares three different methods for the extraction of genomic DNA from formalin-fixed paraffin-embedded (inflammatory fibrous hyperplasia) and non-formalin-fixed (normal oral mucosa) samples: phenol with enzymatic digestion, and silica with and without enzymatic digestion. The amplification of DNA by means of the PCR technique was carried out with primers for the exon 7 of human keratin type 14. Amplicons were analyzed by means of electrophoresis in an 8% polyacrylamide gel with 5% glycerol, followed by silver-staining visualization. The phenol/enzymatic digestion and the silica/enzymatic digestion methods provided amplicons from both tissue samples. The method described is a potential aid in the establishment of the histopathologic diagnosis and in retrospective studies with archival paraffin-embedded samples.

Comparison of hard tissues that are useful for DNA analysis in forensic autopsy.

PubMed

Kaneko, Yu; Ohira, Hiroshi; Tsuda, Yukio; Yamada, Yoshihiro

2015-11-01

Forensic analysis of DNA from hard tissues can be important when investigating a variety of cases resulting from mass disaster or criminal cases. This study was conducted to evaluate the most suitable tissues, method and sample size for processing of hard tissues prior to DNA isolation. We also evaluated the elapsed time after death in relation to the quantity of DNA extracted. Samples of hard tissues (37 teeth, 42 skull, 42 rib, and 39 nails) from 42 individuals aged between 50 and 83 years were used. The samples were taken from remains following forensic autopsy (from 2 days to 2 years after death). To evaluate the integrity of the nuclear DNA isolated, the percentage of allele calls for short tandem repeat profiles were compared between the hard tissues. DNA typing results indicated that until 1 month after death, any of the four hard tissue samples could be used as an alternative to teeth, allowing analysis of all of the loci. However, in terms of the sampling site, collection method and sample size adjustment, the rib appeared to be the best choice in view of the ease of specimen preparation. Our data suggest that the rib could be an alternative hard tissue sample for DNA analysis of human remains. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

PubMed Central

Ramírez, Juan Carlos; Cura, Carolina Inés; Moreira, Otacilio da Cruz; Lages-Silva, Eliane; Juiz, Natalia; Velázquez, Elsa; Ramírez, Juan David; Alberti, Anahí; Pavia, Paula; Flores-Chávez, María Delmans; Muñoz-Calderón, Arturo; Pérez-Morales, Deyanira; Santalla, José; Guedes, Paulo Marcos da Matta; Peneau, Julie; Marcet, Paula; Padilla, Carlos; Cruz-Robles, David; Valencia, Edward; Crisante, Gladys Elena; Greif, Gonzalo; Zulantay, Inés; Costales, Jaime Alfredo; Alvarez-Martínez, Miriam; Martínez, Norma Edith; Villarroel, Rodrigo; Villarroel, Sandro; Sánchez, Zunilda; Bisio, Margarita; Parrado, Rudy; Galvão, Lúcia Maria da Cunha; da Câmara, Antonia Cláudia Jácome; Espinoza, Bertha; de Noya, Belkisyole Alarcón; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G.

2015-01-01

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease. PMID:26320872

Human Papillomavirus (HPV) Infection in Squamous Cell Carcinomas Arising From the Oropharynx: Detection of HPV DNA and p16 Immunohistochemistry as Diagnostic and Prognostic Indicators—A Pilot Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bussu, Francesco, E-mail: francesco.bussu.md@gmail.com; Sali, Michela; Gallus, Roberto

Purpose: Human papillomavirus (HPV) 16 infection is associated with oropharyngeal carcinogenesis and is likely the cause of the reported increase in disease incidence. We evaluated the prevalence of HPV infection and the reliability of different diagnostic tools using primary tumor samples from a cohort of 50 patients. Methods and Materials: Formalin-fixed paraffin-embedded (FFPE) tumor samples were collected from all 50 consecutive primary oropharyngeal SCC patients who were enrolled in the study; fresh tumor samples were available in 22 cases. NucliSENS EasyQ HPVv1 was used for RNA, and Digene Hybrid Capture-2(HC2) was used for DNA detection. p16 Expression was evaluated bymore » immunohistochemistry in FPPE specimens. Results: Based on the DNA detection assay on FFPE samples, the frequency of high-risk HPV infection was 32%. The agreement rate between HPV RNA and HPV DNA detection in fresh samples was 100%. The agreement rate between p16 immunohistochemistry (IHC) and the detection of HPV DNA in the FFPE samples was fair but not excellent (κ = 0.618). HPV DNA detection was highly significant, as measured by disease-specific survival and determined using a Wilcoxon test (P=.001). p16 IHC also exhibited a prognostic value but with a lower statistical significance (P=.0475). The detection of HPV DNA, but not p16 IHC, was also significantly correlated with locoregional control (P=.0461). Conclusion: Diagnostic methods based on the detection of HPV nucleic acids appear to be more reliable and objective because they do not require reading by a trained histopathologist. Furthermore, the detection of HPV DNA exhibits an improved correlation with survival, and therefore appears definitely more reliable than p16 IHC for routine use in clinical practice.« less

Investigating population continuity with ancient DNA under a spatially explicit simulation framework.

PubMed

Silva, Nuno Miguel; Rio, Jeremy; Currat, Mathias

2017-12-15

Recent advances in sequencing technologies have allowed for the retrieval of ancient DNA data (aDNA) from skeletal remains, providing direct genetic snapshots from diverse periods of human prehistory. Comparing samples taken in the same region but at different times, hereafter called "serial samples", may indicate whether there is continuity in the peopling history of that area or whether an immigration of a genetically different population has occurred between the two sampling times. However, the exploration of genetic relationships between serial samples generally ignores their geographical locations and the spatiotemporal dynamics of populations. Here, we present a new coalescent-based, spatially explicit modelling approach to investigate population continuity using aDNA, which includes two fundamental elements neglected in previous methods: population structure and migration. The approach also considers the extensive temporal and geographical variance that is commonly found in aDNA population samples. We first showed that our spatially explicit approach is more conservative than the previous (panmictic) approach and should be preferred to test for population continuity, especially when small and isolated populations are considered. We then applied our method to two mitochondrial datasets from Germany and France, both including modern and ancient lineages dating from the early Neolithic. The results clearly reject population continuity for the maternal line over the last 7500 years for the German dataset but not for the French dataset, suggesting regional heterogeneity in post-Neolithic migratory processes. Here, we demonstrate the benefits of using a spatially explicit method when investigating population continuity with aDNA. It constitutes an improvement over panmictic methods by considering the spatiotemporal dynamics of genetic lineages and the precise location of ancient samples. The method can be used to investigate population continuity between any pair of serial samples (ancient-ancient or ancient-modern) and to investigate more complex evolutionary scenarios. Although we based our study on mitochondrial DNA sequences, diploid molecular markers of different types (DNA, SNP, STR) can also be simulated with our approach. It thus constitutes a promising tool for the analysis of the numerous aDNA datasets being produced, including genome wide data, in humans but also in many other species.

Evaluating droplet digital PCR for the quantification of human genomic DNA: converting copies per nanoliter to nanograms nuclear DNA per microliter.

PubMed

Duewer, David L; Kline, Margaret C; Romsos, Erica L; Toman, Blaza

2018-05-01

The highly multiplexed polymerase chain reaction (PCR) assays used for forensic human identification perform best when used with an accurately determined quantity of input DNA. To help ensure the reliable performance of these assays, we are developing a certified reference material (CRM) for calibrating human genomic DNA working standards. To enable sharing information over time and place, CRMs must provide accurate and stable values that are metrologically traceable to a common reference. We have shown that droplet digital PCR (ddPCR) limiting dilution end-point measurements of the concentration of DNA copies per volume of sample can be traceably linked to the International System of Units (SI). Unlike values assigned using conventional relationships between ultraviolet absorbance and DNA mass concentration, entity-based ddPCR measurements are expected to be stable over time. However, the forensic community expects DNA quantity to be stated in terms of mass concentration rather than entity concentration. The transformation can be accomplished given SI-traceable values and uncertainties for the number of nucleotide bases per human haploid genome equivalent (HHGE) and the average molar mass of a nucleotide monomer in the DNA polymer. This report presents the considerations required to establish the metrological traceability of ddPCR-based mass concentration estimates of human nuclear DNA. Graphical abstract The roots of metrological traceability for human nuclear DNA mass concentration results. Values for the factors in blue must be established experimentally. Values for the factors in red have been established from authoritative source materials. HHGE stands for "haploid human genome equivalent"; there are two HHGE per diploid human genome.

Single-copy gene detection using branched DNA (bDNA) in situ hybridization.

PubMed

Player, A N; Shen, L P; Kenny, D; Antao, V P; Kolberg, J A

2001-05-01

We have developed a branched DNA in situ hybridization (bDNA ISH) method for detection of human papillomavirus (HPV) DNA in whole cells. Using human cervical cancer cell lines with known copies of HPV DNA, we show that the bDNA ISH method is highly sensitive, detecting as few as one or two copies of HPV DNA per cell. By modifying sample pretreatment, viral mRNA or DNA sequences can be detected using the same set of oligonucleotide probes. In experiments performed on mixed populations of cells, the bDNA ISH method is highly specific and can distinguish cells with HPV-16 from cells with HPV-18 DNA. Furthermore, we demonstrate that the bDNA ISH method provides precise localization, yielding positive signals retained within the subcellular compartments in which the target nucleic acid sequences are localized. As an effective and convenient means for nucleic acid detection, the bDNA ISH method is applicable to the detection of cancers and infectious agents. (J Histochem Cytochem 49:603-611, 2001)

Mitochondrial DNA diversity of present-day Aboriginal Australians and implications for human evolution in Oceania.

PubMed

Nagle, Nano; Ballantyne, Kaye N; van Oven, Mannis; Tyler-Smith, Chris; Xue, Yali; Wilcox, Stephen; Wilcox, Leah; Turkalov, Rust; van Oorschot, Roland A H; van Holst Pellekaan, Sheila; Schurr, Theodore G; McAllister, Peter; Williams, Lesley; Kayser, Manfred; Mitchell, R John

2017-03-01

Aboriginal Australians are one of the more poorly studied populations from the standpoint of human evolution and genetic diversity. Thus, to investigate their genetic diversity, the possible date of their ancestors' arrival and their relationships with neighboring populations, we analyzed mitochondrial DNA (mtDNA) diversity in a large sample of Aboriginal Australians. Selected mtDNA single-nucleotide polymorphisms and the hypervariable segment haplotypes were analyzed in 594 Aboriginal Australians drawn from locations across the continent, chiefly from regions not previously sampled. Most (~78%) samples could be assigned to mtDNA haplogroups indigenous to Australia. The indigenous haplogroups were all ancient (with estimated ages >40 000 years) and geographically widespread across the continent. The most common haplogroup was P (44%) followed by S (23%) and M42a (9%). There was some geographic structure at the haplotype level. The estimated ages of the indigenous haplogroups range from 39 000 to 55 000 years, dates that fit well with the estimated date of colonization of Australia based on archeological evidence (~47 000 years ago). The distribution of mtDNA haplogroups in Australia and New Guinea supports the hypothesis that the ancestors of Aboriginal Australians entered Sahul through at least two entry points. The mtDNA data give no support to the hypothesis of secondary gene flow into Australia during the Holocene, but instead suggest long-term isolation of the continent.

A panel of microsatellites to individually identify leopards and its application to leopard monitoring in human dominated landscapes.

PubMed

Mondol, Samrat; Navya, R; Athreya, Vidya; Sunagar, Kartik; Selvaraj, Velu Mani; Ramakrishnan, Uma

2009-12-04

Leopards are the most widely distributed of the large cats, ranging from Africa to the Russian Far East. Because of habitat fragmentation, high human population densities and the inherent adaptability of this species, they now occupy landscapes close to human settlements. As a result, they are the most common species involved in human wildlife conflict in India, necessitating their monitoring. However, their elusive nature makes such monitoring difficult. Recent advances in DNA methods along with non-invasive sampling techniques can be used to monitor populations and individuals across large landscapes including human dominated ones. In this paper, we describe a DNA-based method for leopard individual identification where we used fecal DNA samples to obtain genetic material. Further, we apply our methods to non-invasive samples collected in a human-dominated landscape to estimate the minimum number of leopards in this human-leopard conflict area in Western India. In this study, 25 of the 29 tested cross-specific microsatellite markers showed positive amplification in 37 wild-caught leopards. These loci revealed varied levels of polymorphism (four-12 alleles) and heterozygosity (0.05-0.79). Combining data on amplification success (including non-invasive samples) and locus specific polymorphisms, we showed that eight loci provide a sibling probability of identity of 0.0005, suggesting that this panel can be used to discriminate individuals in the wild. When this microsatellite panel was applied to fecal samples collected from a human-dominated landscape, we identified 7 individuals, with a sibling probability of identity of 0.001. Amplification success of field collected scats was up to 72%, and genotype error ranged from 0-7.4%. Our results demonstrated that the selected panel of eight microsatellite loci can conclusively identify leopards from various kinds of biological samples. Our methods can be used to monitor leopards over small and large landscapes to assess population trends, as well as could be tested for population assignment in forensic applications.

A panel of microsatellites to individually identify leopards and its application to leopard monitoring in human dominated landscapes

PubMed Central

2009-01-01

Background Leopards are the most widely distributed of the large cats, ranging from Africa to the Russian Far East. Because of habitat fragmentation, high human population densities and the inherent adaptability of this species, they now occupy landscapes close to human settlements. As a result, they are the most common species involved in human wildlife conflict in India, necessitating their monitoring. However, their elusive nature makes such monitoring difficult. Recent advances in DNA methods along with non-invasive sampling techniques can be used to monitor populations and individuals across large landscapes including human dominated ones. In this paper, we describe a DNA-based method for leopard individual identification where we used fecal DNA samples to obtain genetic material. Further, we apply our methods to non-invasive samples collected in a human-dominated landscape to estimate the minimum number of leopards in this human-leopard conflict area in Western India. Results In this study, 25 of the 29 tested cross-specific microsatellite markers showed positive amplification in 37 wild-caught leopards. These loci revealed varied levels of polymorphism (four-12 alleles) and heterozygosity (0.05-0.79). Combining data on amplification success (including non-invasive samples) and locus specific polymorphisms, we showed that eight loci provide a sibling probability of identity of 0.0005, suggesting that this panel can be used to discriminate individuals in the wild. When this microsatellite panel was applied to fecal samples collected from a human-dominated landscape, we identified 7 individuals, with a sibling probability of identity of 0.001. Amplification success of field collected scats was up to 72%, and genotype error ranged from 0-7.4%. Conclusion Our results demonstrated that the selected panel of eight microsatellite loci can conclusively identify leopards from various kinds of biological samples. Our methods can be used to monitor leopards over small and large landscapes to assess population trends, as well as could be tested for population assignment in forensic applications. PMID:19961605

Detection of human papillomavirus in pterygium and conjunctival papilloma by hybrid capture II and PCR assays.

PubMed

Takamura, Y; Kubo, E; Tsuzuki, S; Akagi, Y

2008-11-01

To elucidate the putative role of human papillomavirus (HPV) infection in pterygium and conjunctival papilloma. Hybrid capture II (HC-II) and polymerase chain reaction (PCR) assays were performed to detect HPV in pterygium (42 samples obtained from 40 patients) and conjunctival papilloma (8 samples from 6 patients). The amount of HPV DNA was evaluated by measurement of relative light units (RLUs) on a luminometer. All papilloma samples were positive for HPV DNA by PCR and HC-II. The RLU values for specimens of recurrent and re-recurrent papilloma were markedly higher than those for specimens of primary lesions. HPV was detected by PCR in 2 of 42 (4.8%) beta-globin-positive pterygium specimens, whereas HC-II showed that HPV was negative in all pterygium samples. Our results support the hypothesis that HPV DNA is associated with the pathogenesis of conjunctival papilloma, but not pterygium. RLU measurement by HC-II may serve as a marker for evaluating the activity of HPV in conjunctival tumours.

High throughput DNA damage quantification of human tissue with home-based collection device

DOE Office of Scientific and Technical Information (OSTI.GOV)

Costes, Sylvain V.; Tang, Jonathan; Yannone, Steven M.

Kits, methods and systems for providing a service to provide a subject with information regarding the state of a subject's DNA damage. Collection, processing and analysis of samples are also described.

Introducing Human Population Biology through an Easy Laboratory Exercise on Mitochondrial DNA

ERIC Educational Resources Information Center

Pardinas, Antonio F.; Dopico, Eduardo; Roca, Agustin; Garcia-Vazquez, Eva; Lopez, Belen

2010-01-01

This article describes an easy and cheap laboratory exercise for students to discover their own mitochondrial haplogroup. Students use buccal swabs to obtain mucosa cells as noninvasive tissue samples, extract DNA, and with a simple polymerase chain reaction-restriction fragment length polymorphism analysis they can obtain DNA fragments of…

Use of FTA elute card impregnated with cervicovaginal sample directly into the amplification reaction increases the detection of human papillomavirus DNA

PubMed Central

Santos, Carla R.; Franciscatto, Laura G.; Barcellos, Regina B.; Almeida, Sabrina E. M.; Rossetti, Maria Lucia R.

2012-01-01

This study aimed to evaluate the use of the FTA elute cardTM impregnated with cervicovaginal sample directly in the PCR amplification for detection of HPV-DNA. The results were compared to a reference technique. This method was more efficient than the protocol indicated by the manufacturer, identifying 91.7% against 54.2% of the positive samples. PMID:24031844

Validation of a DNA IQ-based extraction method for TECAN robotic liquid handling workstations for processing casework.

PubMed

Frégeau, Chantal J; Lett, C Marc; Fourney, Ron M

2010-10-01

A semi-automated DNA extraction process for casework samples based on the Promega DNA IQ™ system was optimized and validated on TECAN Genesis 150/8 and Freedom EVO robotic liquid handling stations configured with fixed tips and a TECAN TE-Shake™ unit. The use of an orbital shaker during the extraction process promoted efficiency with respect to DNA capture, magnetic bead/DNA complex washes and DNA elution. Validation studies determined the reliability and limitations of this shaker-based process. Reproducibility with regards to DNA yields for the tested robotic workstations proved to be excellent and not significantly different than that offered by the manual phenol/chloroform extraction. DNA extraction of animal:human blood mixtures contaminated with soil demonstrated that a human profile was detectable even in the presence of abundant animal blood. For exhibits containing small amounts of biological material, concordance studies confirmed that DNA yields for this shaker-based extraction process are equivalent or greater to those observed with phenol/chloroform extraction as well as our original validated automated magnetic bead percolation-based extraction process. Our data further supports the increasing use of robotics for the processing of casework samples. Crown Copyright © 2009. Published by Elsevier Ireland Ltd. All rights reserved.

Detecting and Estimating Contamination of Human DNA Samples in Sequencing and Array-Based Genotype Data

PubMed Central

Jun, Goo; Flickinger, Matthew; Hetrick, Kurt N.; Romm, Jane M.; Doheny, Kimberly F.; Abecasis, Gonçalo R.; Boehnke, Michael; Kang, Hyun Min

2012-01-01

DNA sample contamination is a serious problem in DNA sequencing studies and may result in systematic genotype misclassification and false positive associations. Although methods exist to detect and filter out cross-species contamination, few methods to detect within-species sample contamination are available. In this paper, we describe methods to identify within-species DNA sample contamination based on (1) a combination of sequencing reads and array-based genotype data, (2) sequence reads alone, and (3) array-based genotype data alone. Analysis of sequencing reads allows contamination detection after sequence data is generated but prior to variant calling; analysis of array-based genotype data allows contamination detection prior to generation of costly sequence data. Through a combination of analysis of in silico and experimentally contaminated samples, we show that our methods can reliably detect and estimate levels of contamination as low as 1%. We evaluate the impact of DNA contamination on genotype accuracy and propose effective strategies to screen for and prevent DNA contamination in sequencing studies. PMID:23103226

Genetic determinants of freckle occurrence in the Spanish population: Towards ephelides prediction from human DNA samples.

PubMed

Hernando, Barbara; Ibañez, Maria Victoria; Deserio-Cuesta, Julio Alberto; Soria-Navarro, Raquel; Vilar-Sastre, Inca; Martinez-Cadenas, Conrado

2018-03-01

Prediction of human pigmentation traits, one of the most differentiable externally visible characteristics among individuals, from biological samples represents a useful tool in the field of forensic DNA phenotyping. In spite of freckling being a relatively common pigmentation characteristic in Europeans, little is known about the genetic basis of this largely genetically determined phenotype in southern European populations. In this work, we explored the predictive capacity of eight freckle and sunlight sensitivity-related genes in 458 individuals (266 non-freckled controls and 192 freckled cases) from Spain. Four loci were associated with freckling (MC1R, IRF4, ASIP and BNC2), and female sex was also found to be a predictive factor for having a freckling phenotype in our population. After identifying the most informative genetic variants responsible for human ephelides occurrence in our sample set, we developed a DNA-based freckle prediction model using a multivariate regression approach. Once developed, the capabilities of the prediction model were tested by a repeated 10-fold cross-validation approach. The proportion of correctly predicted individuals using the DNA-based freckle prediction model was 74.13%. The implementation of sex into the DNA-based freckle prediction model slightly improved the overall prediction accuracy by 2.19% (76.32%). Further evaluation of the newly-generated prediction model was performed by assessing the model's performance in a new cohort of 212 Spanish individuals, reaching a classification success rate of 74.61%. Validation of this prediction model may be carried out in larger populations, including samples from different European populations. Further research to validate and improve this newly-generated freckle prediction model will be needed before its forensic application. Together with DNA tests already validated for eye and hair colour prediction, this freckle prediction model may lead to a substantially more detailed physical description of unknown individuals from DNA found at the crime scene. Copyright © 2017 Elsevier B.V. All rights reserved.

The role of human papilloma virus and herpes viruses in the etiology of nasal polyposis.

PubMed

Koçoğlu, Mücahide Esra; Mengeloğlu, Fırat Zafer; Apuhan, Tayfun; Özsoy, Şeyda; Yilmaz, Beyhan

2016-02-17

The aim of this study was to investigate the etiological role of human papilloma virus (HPV), herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpes virus-6 (HHV-6) and -7 (HHV-7) in the occurrence of nasal polyposis. Nasal polyp samples from 30 patients with nasal polyposis and normal nasal mucosa from 10 patients without nasal polyps were obtained. DNA was extracted from tissues. Real-time polymerase chain reaction was performed for all runs. No HSV-1, HSV-2, or VZV was detected in the samples. Among the patient samples, EBV and HHV-7 DNA were detected in 18 (60%), HHV-6 was detected in 20 (66.7%), and HPV was detected in 4 (13.3%) samples. Among the controls, CMV DNA was positive in one (10%). EBV was positive in 5 (50%), HHV-6 and HHV-7 were positive in 7 (70%), and HPV was positive in 2 (20%) samples. No significant difference was found among the groups with any test in terms of positivity. The association of Herpesviridae and HPV with the pathogenesis of nasal polyps was investigated in this study and no relationship was found. Thus, these viruses do not play a significant role in the formation of nasal polyps.

Real-time PCR assays using internal controls for quantitation of HPV-16 and beta-globin DNA in cervicovaginal lavages.

PubMed

Lefevre, Jonas; Hankins, Catherine; Pourreaux, Karina; Voyer, Hélène; Coutlée, François

2003-12-01

High-risk human papillomavirus 16 (HPV-16) DNA viral load has been measured with real-time PCR assays by amplifying HPV-16 and a human gene. However, these assays have not used internal controls (ICs) to screen for the presence of inhibitors contained in samples. To quantitate HPV-16 DNA and cell content with real-time PCR, ICs for HPV-16 DNA and beta-globin were synthesised and used to control for inhibition. The assays were sensitive and linear over 5 logs. Good reproducibility was achieved with inter-run coefficients of variation of 23% (10(2) HPV-16 copies), 12% (10(4) HPV-16 copies), 17% (274 beta-globin DNA copies) and 7% (27,400 beta-globin DNA copies). Samples containing 56,800,000, 306,000, 18,000, and 4,070 HPV-16 copies/microg of cellular DNA were tested blindly and estimated to contain 48,800,000, 479,000, 20,300, and 6,620 HPV-16 copies/microg of DNA (mean ratio of measured to expected viral load of 1.27+/-0.32). Inhibition of amplification of HPV-16 and beta-globin ICs by six samples known to contain PCR inhibitors was variable: four inhibited both ICs while two inhibited only the HPV-16 IC. The use of internal controls with real-time PCR for HPV-16 quantitation allows to screen for the presence of inhibitors that do not affect equally primer-driven genomic amplification.

Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA[reg])

DOE Office of Scientific and Technical Information (OSTI.GOV)

Evenson, Donald P.; Wixon, Regina

Studies over the past two decades have clearly shown that reproductive toxicants cause sperm DNA fragmentation. This DNA fragmentation can usually be detected prior to observing alterations of metaphase chromosomes in embryos. Thus, Sperm Chromatin Structure Assay (SCSA)-detected DNA damage is viewed as the molecular precursor to later gross chromosome damage observed under the light microscope. SCSA measurements of animal or human sperm consist of first obtaining a fresh or flash frozen neat semen sample in LN2 or dry ice. Samples are then sent to a SCSA diagnostic laboratory where the samples are thawed, diluted to {approx}1-2 x 106 sperm/ml,more » treated for 30 s with a pH 1.2 detergent buffer and then stained with acridine orange (AO). The low pH partially denatures DNA at the sites of DNA strand breaks and the AO-ssDNA fluoresces red while the AO-dsDNA fluoresces green. Flow cytometry measurements of 5000 sperm/sample provide statistically robust data on the ratio of red to green sperm, the extent of the DNA fragmentation and the standard deviations of measures. Numerous experiments on rodents treated with reproductive toxicants clearly showed that SCSA measures are highly dose responsive and have a very low CV. Different agents that act on germ cells at various stages of development usually showed sperm DNA fragmentation when that germ cell fraction arrived in the epididymis or ejaculate. Some of these treated samples were capable of successful in vitro fertilization but with frequent embryo failure. A 2-year longitudinal study of men living a valley town with a reported abnormal level of infertility and spontaneous miscarriages and also a seasonal atmospheric smog pollution, showed, for the first time, that SCSA measurements of human sperm DNA fragmentation were detectable and correlated with dosage of air pollution while the classical semen measures were not correlated. Also, young men spraying pesticides without protective gear are at an increased risk for elevated sperm DNA fragmentation. Extensive DNA fragmentation probably cannot be repaired by the egg and the spontaneous abortion rate is {approx}2x higher if a man has more than 30% of sperm showing DNA fragmentation. DNA fragmentation is an excellent marker for exposure to potential reproductive toxicants and a diagnostic/prognostic tool for potential male infertility.« less

Ancient pathogen DNA in archaeological samples detected with a Microbial Detection Array.

PubMed

Devault, Alison M; McLoughlin, Kevin; Jaing, Crystal; Gardner, Shea; Porter, Teresita M; Enk, Jacob M; Thissen, James; Allen, Jonathan; Borucki, Monica; DeWitte, Sharon N; Dhody, Anna N; Poinar, Hendrik N

2014-03-06

Ancient human remains of paleopathological interest typically contain highly degraded DNA in which pathogenic taxa are often minority components, making sequence-based metagenomic characterization costly. Microarrays may hold a potential solution to these challenges, offering a rapid, affordable, and highly informative snapshot of microbial diversity in complex samples without the lengthy analysis and/or high cost associated with high-throughput sequencing. Their versatility is well established for modern clinical specimens, but they have yet to be applied to ancient remains. Here we report bacterial profiles of archaeological and historical human remains using the Lawrence Livermore Microbial Detection Array (LLMDA). The array successfully identified previously-verified bacterial human pathogens, including Vibrio cholerae (cholera) in a 19th century intestinal specimen and Yersinia pestis ("Black Death" plague) in a medieval tooth, which represented only minute fractions (0.03% and 0.08% alignable high-throughput shotgun sequencing reads) of their respective DNA content. This demonstrates that the LLMDA can identify primary and/or co-infecting bacterial pathogens in ancient samples, thereby serving as a rapid and inexpensive paleopathological screening tool to study health across both space and time.

Microbial Communities in Pre-Columbian Coprolites

PubMed Central

Santiago-Rodriguez, Tasha M.; Narganes-Storde, Yvonne M.; Chanlatte, Luis; Crespo-Torres, Edwin; Toranzos, Gary A.; Jimenez-Flores, Rafael; Hamrick, Alice; Cano, Raul J.

2013-01-01

The study of coprolites from earlier cultures represents a great opportunity to study an “unaltered” composition of the intestinal microbiota. To test this, pre-Columbian coprolites from two cultures, the Huecoid and Saladoid, were evaluated for the presence of DNA, proteins and lipids by cytochemical staining, human and/or dog-specific Bacteroides spp. by PCR, as well as bacteria, fungi and archaea using Terminal Restriction Fragment analyses. DNA, proteins and lipids, and human-specific Bacteroides DNA were detected in all coprolites. Multidimensional scaling analyses resulted in spatial arrangements of microbial profiles by culture, further supported by cluster analysis and ANOSIM. Differences between the microbial communities were positively correlated with culture, and SIMPER analysis indicated 68.8% dissimilarity between the Huecoid and Saladoid. Proteobacteria, Bacteroidetes and methanogens were found in all coprolite samples. Propionebacteria, Shewanella and lactic acid bacteria dominated in the Huecoid samples, while Acidobacteria, and peptococci were dominant in Saladoid samples. Yeasts, including Candida albicans and Crypotococcus spp. were found in all samples. Basidiomycetes were the most notable fungi in Huecoid samples while Ascomycetes predominated in Saladoid samples, suggesting differences in dietary habits. Our study provides an approach for the study of the microbial communities of coprolite samples from various cultures. PMID:23755194

Authentication of forensic DNA samples.

PubMed

Frumkin, Dan; Wasserstrom, Adam; Davidson, Ariane; Grafit, Arnon

2010-02-01

Over the past twenty years, DNA analysis has revolutionized forensic science, and has become a dominant tool in law enforcement. Today, DNA evidence is key to the conviction or exoneration of suspects of various types of crime, from theft to rape and murder. However, the disturbing possibility that DNA evidence can be faked has been overlooked. It turns out that standard molecular biology techniques such as PCR, molecular cloning, and recently developed whole genome amplification (WGA), enable anyone with basic equipment and know-how to produce practically unlimited amounts of in vitro synthesized (artificial) DNA with any desired genetic profile. This artificial DNA can then be applied to surfaces of objects or incorporated into genuine human tissues and planted in crime scenes. Here we show that the current forensic procedure fails to distinguish between such samples of blood, saliva, and touched surfaces with artificial DNA, and corresponding samples with in vivo generated (natural) DNA. Furthermore, genotyping of both artificial and natural samples with Profiler Plus((R)) yielded full profiles with no anomalies. In order to effectively deal with this problem, we developed an authentication assay, which distinguishes between natural and artificial DNA based on methylation analysis of a set of genomic loci: in natural DNA, some loci are methylated and others are unmethylated, while in artificial DNA all loci are unmethylated. The assay was tested on natural and artificial samples of blood, saliva, and touched surfaces, with complete success. Adopting an authentication assay for casework samples as part of the forensic procedure is necessary for maintaining the high credibility of DNA evidence in the judiciary system.

Trypanosoma cruzi diversity in the Gran Chaco: mixed infections and differential host distribution of TcV and TcVI.

PubMed

Monje-Rumi, María M; Brandán, Cecilia Pérez; Ragone, Paula G; Tomasini, Nicolás; Lauthier, Juan J; Alberti D'Amato, Anahí M; Cimino, Rubén O; Orellana, Viviana; Basombrío, Miguel A; Diosque, Patricio

2015-01-01

The transmission cycles of Trypanosoma cruzi in the Gran Chaco are complex networks involving domestic and wild components, whose interrelationships are not well understood. Knowing the circuit of transmission of the different Discrete Typing Units (DTUs) of T. cruzi in the complex environment of the Chaco region is relevant to understanding how the different components (reservoirs, vectors, ecotopes) interact. In the present study we identified the DTUs infecting humans and dogs in two rural areas of the Gran Chaco in Argentina, using molecular methods which avoid parasite culture. Blood samples of humans and dogs were typified by PCR-DNA blotting and hybridization assays with five specific DNA probes (TcI, TcII, TcIII, TcV and TcVI). PCR analyses were performed on seropositive human and dog samples and showed the presence of T. cruzi DNA in 41.7% (98/235) and 53% (35/66) samples, respectively. The identification of infective DTUs was determined in 83.6% (82/98) and 91.4% (32/35) in human and dog samples, respectively. Single infections (36.7% - 36/98) and a previously not detected high proportion of mixed infections (47.9% - 47/98) were found. In a 15.3% (15/98) of samples the infecting DTU was not identified. Among the single infections TcV was the most prevalent DTU (30.6% - 30/98) in human samples; while TcVI (42.8% - 15/35) showed the highest prevalence in dog samples. TcV/TcVI was the most prevalent mixed infection in humans (32.6% - 32/98); and TcI/TcVI (14.3% - 5/35) in dogs. Significant associations between TcV with humans and TcVI with dogs were detected. For the first time, the presence of TcIII was detected in humans from this region. The occurrence of one human infected whit TcIII (a principally wild DTU) could be suggested the emergence of this, in domestic cycles in the Gran Chaco. Copyright © 2014 Elsevier B.V. All rights reserved.

High Prevalence of Human Parvovirus B19 DNA in Myocardial Autopsy Samples from Subjects without Myocarditis or Dilative Cardiomyopathy▿

PubMed Central

Schenk, Thomas; Enders, Martin; Pollak, Stefan; Hahn, Ralph; Huzly, Daniela

2009-01-01

Human parvovirus B19 has been linked to a variety of cardiac diseases, as well as to erythema infectiosum, acute arthropathy, and fetal hydrops. A causal association between viral infection and cardiac disease was frequently postulated following the detection of B19 DNA by PCR in endomyocardial biopsy specimens. Since the lifelong persistence of B19 DNA in bone marrow, skin, synovia, tonsils, and liver was previously reported, the aim of our study was to investigate the possibility of asymptomatic B19 DNA persistence in heart tissue. Myocardial autopsy and postmortem blood samples were prospectively collected from 69 bodies sent to the Department of Forensic Medicine, Freiburg University Medical Center, for inquests. All study subjects were screened for B19-specific antibodies using a commercial enzyme immunoassay. Tissue samples were analyzed by real-time PCR for the presence of viral DNA. Since the presence of B19 genotype 2, known to have been circulating before 1960, would prove long-lasting persistence, the presence of the B19 genotype was retrospectively determined in seven of the study subjects by melting temperature analysis and sequencing of the PCR product. B19 DNA was found in myocardial samples from 46 of 48 seropositive and in none of 21 seronegative individuals. B19 genotype 1 was found in three patients born between 1950 and 1969. Genotype 2 was found in four patients born between 1927 and 1957. Our findings suggest lifelong persistence of B19 DNA in heart tissue. Thus, the detection of B19 DNA in myocardial biopsy specimens alone is not sufficient to postulate a relationship between B19 infection and cardiac disease. PMID:19005147

Development of internal amplification controls for DNA profiling with the AmpFℓSTR(®) SGM Plus(®) kit.

PubMed

Zahra, Nathalie; Hadi, Sibte; Smith, Judith A; Iyengar, Arati; Goodwin, William

2011-06-01

DNA extracted from forensic samples can be degraded and also contain co-extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpFℓSTR® SGM Plus® kit. The co-amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC₉₀ and IAC₄₁₀) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX-labelled primers. Co-amplification of IAC₉₀ and IAC₄₁₀ was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC₉₀ and IAC₄₁₀ were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC₉₀ signal was still present after all human DNA loci fail to amplify; in contrast, the IAC₄₁₀ signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples.

PubMed

Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gébel, Gabriella; Röscheisen, Christiane

2012-09-01

Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Rapid quantification and sex determination of forensic evidence materials.

PubMed

Andréasson, Hanna; Allen, Marie

2003-11-01

DNA quantification of forensic evidence is very valuable for an optimal use of the available biological material. Moreover, sex determination is of great importance as additional information in criminal investigations as well as in identification of missing persons, no suspect cases, and ancient DNA studies. While routine forensic DNA analysis based on short tandem repeat markers includes a marker for sex determination, analysis of samples containing scarce amounts of DNA is often based on mitochondrial DNA, and sex determination is not performed. In order to allow quantification and simultaneous sex determination on minute amounts of DNA, an assay based on real-time PCR analysis of a marker within the human amelogenin gene has been developed. The sex determination is based on melting curve analysis, while an externally standardized kinetic analysis allows quantification of the nuclear DNA copy number in the sample. This real-time DNA quantification assay has proven to be highly sensitive, enabling quantification of single DNA copies. Although certain limitations were apparent, the system is a rapid, cost-effective, and flexible assay for analysis of forensic casework samples.

High-throughput detection of human papillomavirus-18 L1 gene methylation, a candidate biomarker for the progression of cervical neoplasia.

PubMed

Turan, Tolga; Kalantari, Mina; Cuschieri, Kate; Cubie, Heather A; Skomedal, Hanne; Bernard, Hans-Ulrich

2007-04-25

The L1 gene of human papillomavirus-18 (HPV-18) is consistently hypermethylated in cervical carcinomas, but frequently hypo- or unmethylated in exfoliated cells from asymptomatic patients. In precancerous lesions, L1 is sporadically hypermethylated, correlating with the severity of the neoplasia. In order to explore the potential of using L1 methylation as a workable biomarker for carcinogenic progression of HPV-18 infections in routinely taken samples, our aim was to develop methylation-detection techniques that were sensitive and rapid without being overly complex technically. Therein, we developed a methylation-specific PCR (MSP) through the design of primer sets that specifically amplify either methylated or unmethylated HPV-18 L1 DNA within bisulfite-modified sample DNA. Amplification of unmethylated and in vitro methylated HPV-18 DNA by MSP resulted in 2500 copies of either of the two L1 DNA species being detected, a satisfactory sensitivity considering that bisulfite treatment leads to the fragmentation of about 99% of sample DNA. The primers proved specific and did not generate false positive results at concentrations exceeding the lowest limit of detection by a factor of 400. DNA from carcinomas yielded PCR signals only with the methylation-specific primers, and not with primers specific for unmethylated L1 genes. The inverse result was obtained with DNA from precursor lesions that contained only hypomethylated DNA. High-grade precursor lesions and carcinomas that contained hyper- as well as hypomethylated L1 DNA yielded PCR signals with both primers. By developing a fluorescence based real-time PCR, we quantitatively analyzed samples with in vitro methylated and unmethylated L1 DNA, and could distinguish clinical samples with hyper- and hypomethylated DNA or mixtures of both DNAs. The methylation-specific and real-time PCR techniques permitted efficient HPV-18 L1 methylation analyses and open the door for larger-scale clinical studies where the utility of methylation status to predict the progression of HPV-18 infection and HPV-18 associated lesions is assessed.

Sequence-Level Mechanisms of Human Epigenome Evolution

PubMed Central

Prendergast, James G.D.; Chambers, Emily V.; Semple, Colin A.M.

2014-01-01

DNA methylation and chromatin states play key roles in development and disease. However, the extent of recent evolutionary divergence in the human epigenome and the influential factors that have shaped it are poorly understood. To determine the links between genome sequence and human epigenome evolution, we examined the divergence of DNA methylation and chromatin states following segmental duplication events in the human lineage. Chromatin and DNA methylation states were found to have been generally well conserved following a duplication event, with the evolution of the epigenome largely uncoupled from the total number of genetic changes in the surrounding DNA sequence. However, the epigenome at tissue-specific, distal regulatory regions was observed to be unusually prone to diverge following duplication, with particular sequence differences, altering known sequence motifs, found to be associated with divergence in patterns of DNA methylation and chromatin. Alu elements were found to have played a particularly prominent role in shaping human epigenome evolution, and we show that human-specific AluY insertion events are strongly linked to the evolution of the DNA methylation landscape and gene expression levels, including at key neurological genes in the human brain. Studying paralogous regions within the same sample enables the study of the links between genome and epigenome evolution while controlling for biological and technical variation. We show DNA methylation and chromatin divergence between duplicated regions are linked to the divergence of particular genetic motifs, with Alu elements having played a disproportionate role in the evolution of the epigenome in the human lineage. PMID:24966180

Ancient DNA studies: new perspectives on old samples

PubMed Central

2012-01-01

In spite of past controversies, the field of ancient DNA is now a reliable research area due to recent methodological improvements. A series of recent large-scale studies have revealed the true potential of ancient DNA samples to study the processes of evolution and to test models and assumptions commonly used to reconstruct patterns of evolution and to analyze population genetics and palaeoecological changes. Recent advances in DNA technologies, such as next-generation sequencing make it possible to recover DNA information from archaeological and paleontological remains allowing us to go back in time and study the genetic relationships between extinct organisms and their contemporary relatives. With the next-generation sequencing methodologies, DNA sequences can be retrieved even from samples (for example human remains) for which the technical pitfalls of classical methodologies required stringent criteria to guaranty the reliability of the results. In this paper, we review the methodologies applied to ancient DNA analysis and the perspectives that next-generation sequencing applications provide in this field. PMID:22697611

Highly sensitive detection of human IgG using a novel bio-barcode assay combined with DNA chip technology

NASA Astrophysics Data System (ADS)

Liu, Zhenbao; Zhou, Bo; Wang, Haiqing; Lu, Feng; Liu, Tianjun; Song, Cunxian; Leng, Xigang

2013-09-01

A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 μm in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL-1 to 10 ng mL-1. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples.

[Individual identification of cadaver parts after a bomb explosion using oligonucleotide fingerprinting by (GTG)5].

PubMed

Kondo, T; Ohshima, T

1998-01-01

A blind shell suddenly and unexpectedly exploded, and 20 dismembered human remains were discovered. DNA fingerprint was performed to determine whether the 20 human remains were derived from one person or not. DNA was isolated from each of the remains and digested by the restriction enzyme Hinf I and Hae III and hybridized with the oligonucleotide probe (GTG)5. DNA fingerprint using Hinf I demonstrated the same band pattern in 17 out of the 20 remains. However, in the remaining 3 samples, two novel strange bands were observed. DNA fingerprint using Hae III showed completely identical pattern in all of the remains.

Asymmetric segregation of template DNA strands in basal-like human breast cancer cell lines

PubMed Central

2013-01-01

Background and methods Stem or progenitor cells from healthy tissues have the capacity to co-segregate their template DNA strands during mitosis. Here, we set out to test whether breast cancer cell lines also possess the ability to asymmetrically segregate their template DNA strands via non-random chromosome co-segregation, and whether this ability correlates with certain properties attributed to breast cancer stem cells (CSCs). We quantified the frequency of asymmetric segregation of template DNA strands in 12 human breast cancer cell lines, and correlated the frequency to molecular subtype, CD44+/CD24-/lo phenotype, and invasion/migration ability. We tested if co-culture with human mesenchymal stem cells, which are known to increase self-renewal, can alter the frequency of asymmetric segregation of template DNA in breast cancer. Results We found a positive correlation between asymmetric segregation of template DNA and the breast cancer basal-like and claudin-low subtypes. There was an inverse correlation between asymmetric segregation of template DNA and Her2 expression. Breast cancer samples with evidence of asymmetric segregation of template DNA had significantly increased invasion and borderline significantly increased migration abilities. Samples with high CD44+/CD24-/lo surface expression were more likely to harbor a consistent population of cells that asymmetrically segregated its template DNA; however, symmetric self-renewal was enriched in the CD44+/CD24-/lo population. Co-culturing breast cancer cells with human mesenchymal stem cells expanded the breast CSC pool and decreased the frequency of asymmetric segregation of template DNA. Conclusions Breast cancer cells within the basal-like subtype can asymmetrically segregate their template DNA strands through non-random chromosome segregation. The frequency of asymmetric segregation of template DNA can be modulated by external factors that influence expansion or self-renewal of CSC populations. Future studies to uncover the underlying mechanisms driving asymmetric segregation of template DNA and dictating cell fate at the time of cell division may explain how CSCs are maintained in tumors. PMID:24238140

Forensic DNA testing.

PubMed

Butler, John M

2011-12-01

Forensic DNA testing has a number of applications, including parentage testing, identifying human remains from natural or man-made disasters or terrorist attacks, and solving crimes. This article provides background information followed by an overview of the process of forensic DNA testing, including sample collection, DNA extraction, PCR amplification, short tandem repeat (STR) allele separation and sizing, typing and profile interpretation, statistical analysis, and quality assurance. The article concludes with discussions of possible problems with the data and other forensic DNA testing techniques.

Effective removal of co-purified inhibitors from extracted DNA samples using synchronous coefficient of drag alteration (SCODA) technology.

PubMed

Schmedes, Sarah; Marshall, Pamela; King, Jonathan L; Budowle, Bruce

2013-07-01

Various types of biological samples present challenges for extraction of DNA suitable for subsequent molecular analyses. Commonly used extraction methods, such as silica membrane columns and phenol-chloroform, while highly successful may still fail to provide a sufficiently pure DNA extract with some samples. Synchronous coefficient of drag alteration (SCODA), implemented in Boreal Genomics' Aurora Nucleic Acid Extraction System (Boreal Genomics, Vancouver, BC), is a new technology that offers the potential to remove inhibitors effectively while simultaneously concentrating DNA. In this initial study, SCODA was tested for its ability to remove various concentrations of forensically and medically relevant polymerase chain reaction (PCR) inhibitors naturally found in tissue, hair, blood, plant, and soil samples. SCODA was used to purify and concentrate DNA from intentionally contaminated DNA samples containing known concentrations of hematin, humic acid, melanin, and tannic acid. The internal positive control (IPC) provided in the Quantifiler™ Human DNA Quantification Kit (Life Technologies, Foster City, CA) and short tandem repeat (STR) profiling (AmpFℓSTR® Identifiler® Plus PCR Amplification Kit; Life Technologies, Foster City, CA) were used to measure inhibition effects and hence purification. SCODA methodology yielded overall higher efficiency of purification of highly contaminated samples compared with the QIAquick® PCR Purification Kit (Qiagen, Valencia, CA). SCODA-purified DNA yielded no cycle shift of the IPC for each sample and yielded greater allele percentage recovery and relative fluorescence unit values compared with the QIAquick® purification method. The Aurora provided an automated, minimal-step approach to successfully remove inhibitors and concentrate DNA from challenged samples.

DNA methylation assessment from human slow- and fast-twitch skeletal muscle fibers

PubMed Central

Begue, Gwénaëlle; Raue, Ulrika; Jemiolo, Bozena

2017-01-01

A new application of the reduced representation bisulfite sequencing method was developed using low-DNA input to investigate the epigenetic profile of human slow- and fast-twitch skeletal muscle fibers. Successful library construction was completed with as little as 15 ng of DNA, and high-quality sequencing data were obtained with 32 ng of DNA. Analysis identified 143,160 differentially methylated CpG sites across 14,046 genes. In both fiber types, selected genes predominantly expressed in slow or fast fibers were hypomethylated, which was supported by the RNA-sequencing analysis. These are the first fiber type-specific methylation data from human skeletal muscle and provide a unique platform for future research. NEW & NOTEWORTHY This study validates a low-DNA input reduced representation bisulfite sequencing method for human muscle biopsy samples to investigate the methylation patterns at a fiber type-specific level. These are the first fiber type-specific methylation data reported from human skeletal muscle and thus provide initial insight into basal state differences in myosin heavy chain I and IIa muscle fibers among young, healthy men. PMID:28057818

Assessing DNA recovery from chewing gum.

PubMed

Eychner, Alison M; Schott, Kelly M; Elkins, Kelly M

2017-01-01

The purpose of this study was to evaluate which DNA extraction method yields the highest quantity of DNA from chewing gum. In this study, several popular extraction methods were tested, including Chelex-100, phenol-chloroform-isoamyl alcohol (PCIA), DNA IQ, PrepFiler, and QIAamp Investigator, and the quantity of DNA recovered from chewing gum was determined using real-time polymerase chain reaction with Quantifiler. Chewed gum control samples were submitted by anonymous healthy adult donors, and discarded environmental chewing gum samples simulating forensic evidence were collected from outside public areas (e.g., campus bus stops, streets, and sidewalks). As expected, results indicate that all methods tested yielded sufficient amplifiable human DNA from chewing gum using the wet-swab method. The QIAamp performed best when DNA was extracted from whole pieces of control gum (142.7 ng on average), and the DNA IQ method performed best on the environmental whole gum samples (29.0 ng on average). On average, the QIAamp kit also recovered the most DNA from saliva swabs. The PCIA method demonstrated the highest yield with wet swabs of the environmental gum (26.4 ng of DNA on average). However, this method should be avoided with whole gum samples (no DNA yield) due to the action of the organic reagents in dissolving and softening the gum and inhibiting DNA recovery during the extraction.

Comparative Assessment of a Self-sampling Device and Gynecologist Sampling for Cytology and HPV DNA Detection in a Rural and Low Resource Setting: Malaysian Experience.

PubMed

Latiff, Latiffah A; Ibrahim, Zaidah; Pei, Chong Pei; Rahman, Sabariah Abdul; Akhtari-Zavare, Mehrnoosh

2015-01-01

This study was conducted to assess the agreement and differences between cervical self-sampling with a Kato device (KSSD) and gynecologist sampling for Pap cytology and human papillomavirus DNA (HPV DNA) detection. Women underwent self-sampling followed by gynecologist sampling during screening at two primary health clinics. Pap cytology of cervical specimens was evaluated for specimen adequacy, presence of endocervical cells or transformation zone cells and cytological interpretation for cells abnormalities. Cervical specimens were also extracted and tested for HPV DNA detection. Positive HPV smears underwent gene sequencing and HPV genotyping by referring to the online NCBI gene bank. Results were compared between samplings by Kappa agreement and McNemar test. For Pap specimen adequacy, KSSD showed 100% agreement with gynecologist sampling but had only 32.3% agreement for presence of endocervical cells. Both sampling showed 100% agreement with only 1 case detected HSIL favouring CIN2 for cytology result. HPV DNA detection showed 86.2%agreement (K=0.64, 95% CI 0.524-0.756, p=0.001) between samplings. KSSD and gynaecologist sampling identified high risk HPV in 17.3% and 23.9% respectively (p= 0.014). The self-sampling using Kato device can serve as a tool in Pap cytology and HPV DNA detection in low resource settings in Malaysia. Self-sampling devices such as KSSD can be used as an alternative technique to gynaecologist sampling for cervical cancer screening among rural populations in Malaysia.

Genetic mutation analysis of human gastric adenocarcinomas using ion torrent sequencing platform.

PubMed

Xu, Zhi; Huo, Xinying; Ye, Hua; Tang, Chuanning; Nandakumar, Vijayalakshmi; Lou, Feng; Zhang, Dandan; Dong, Haichao; Sun, Hong; Jiang, Shouwen; Zhang, Guangchun; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; He, Yan; Yan, Chaowei; Wang, Lu; Su, Ziyi; Li, Yangyang; Gu, Dongying; Zhang, Xiaojing; Wu, Xiaomin; Wei, Xiaowei; Hong, Lingzhi; Zhang, Yangmei; Yang, Jinsong; Gong, Yonglin; Tang, Cuiju; Jones, Lindsey; Huang, Xue F; Chen, Si-Yi; Chen, Jinfei

2014-01-01

Gastric cancer is the one of the major causes of cancer-related death, especially in Asia. Gastric adenocarcinoma, the most common type of gastric cancer, is heterogeneous and its incidence and cause varies widely with geographical regions, gender, ethnicity, and diet. Since unique mutations have been observed in individual human cancer samples, identification and characterization of the molecular alterations underlying individual gastric adenocarcinomas is a critical step for developing more effective, personalized therapies. Until recently, identifying genetic mutations on an individual basis by DNA sequencing remained a daunting task. Recent advances in new next-generation DNA sequencing technologies, such as the semiconductor-based Ion Torrent sequencing platform, makes DNA sequencing cheaper, faster, and more reliable. In this study, we aim to identify genetic mutations in the genes which are targeted by drugs in clinical use or are under development in individual human gastric adenocarcinoma samples using Ion Torrent sequencing. We sequenced 737 loci from 45 cancer-related genes in 238 human gastric adenocarcinoma samples using the Ion Torrent Ampliseq Cancer Panel. The sequencing analysis revealed a high occurrence of mutations along the TP53 locus (9.7%) in our sample set. Thus, this study indicates the utility of a cost and time efficient tool such as Ion Torrent sequencing to screen cancer mutations for the development of personalized cancer therapy.

Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes.

PubMed

Segers-Nolten, G M J; Wyman, C; Wijgers, N; Vermeulen, W; Lenferink, A T M; Hoeijmakers, J H J; Greve, J; Otto, C

2002-11-01

We used scanning confocal fluorescence microscopy to observe and analyze individual DNA- protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measurements were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level approximately 10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level.

Human herpesvirus-6 and -7 DNA in cerebrospinal fluid of facial palsy patients.

PubMed

Kanerva, Mervi; Jääskeläinen, Anne J; Suvela, Minna; Piiparinen, Heli; Vaheri, Antti; Pitkäranta, Anne

2008-04-01

Finding human herpesvirus (HHV)-7 and dual HHV-6A and -6B DNA in cerebrospinal fluid (CSF) of two facial palsy (FP) patients is intriguing but does not allow etiologic conclusions as such. HHV-6 or -7 DNA was revealed in 10% of the CSF samples tested from 70 immunocompetent adolescents and adults; a highly unusual result. How these findings are associated with the diseases they accompany remains to be defined. To determine whether herpes simplex virus (HSV)-1 and -2, varicella-zoster virus (VZV), HHV-6A, -6B, and -7, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) DNA could be found in CSF of FP patients or controls. In all, 33 peripheral FP patients (26 idiopathic, 5 with herpesvirus infection, 1 puerperal, 1 Melkersson-Rosenthal syndrome) (34 CSF samples) and 36 controls (16 nonidiopathic FP, 7 hearing loss, 6 vertigo, 5 headache, 2 other) previously tested for HSV-1, VZV, and HHV-6 DNA by polymerase chain reaction (PCR) were tested with highly sensitive multiplex-PCR and an oligonucleotide microarray method. One FP patient had HHV-7 DNA and another had HHV-6A and -6B DNA simultaneously. In the control group, one HHV-7, one HHV-6A, and three HHV-6B DNA-positive specimens were found.

Enhanced Genetic Analysis of Single Human Bioparticles Recovered by Simplified Micromanipulation from Forensic ‘Touch DNA’ Evidence

PubMed Central

Farash, Katherine; Hanson, Erin K.; Ballantyne, Jack

2015-01-01

DNA profiles can be obtained from ‘touch DNA’ evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a ‘blind-swabbing’ approach will co-sample cellular material from the different individuals, even if the individuals’ cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim’s DNA may be found in significant excess thus masking any potential perpetrator’s DNA. In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, ‘smart analysis’ method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 µl microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., “clumps”) bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material. PMID:25867046

The Rapid-Heat LAMPellet Method: A Potential Diagnostic Method for Human Urogenital Schistosomiasis

PubMed Central

Carranza-Rodríguez, Cristina; Pérez-Arellano, José Luis; Vicente, Belén; López-Abán, Julio; Muro, Antonio

2015-01-01

Background Urogenital schistosomiasis due to Schistosoma haematobium is a serious underestimated public health problem affecting 112 million people - particularly in sub-Saharan Africa. Microscopic examination of urine samples to detect parasite eggs still remains as definitive diagnosis. This work was focussed on developing a novel loop-mediated isothermal amplification (LAMP) assay for detection of S. haematobium DNA in human urine samples as a high-throughput, simple, accurate and affordable diagnostic tool to use in diagnosis of urogenital schistosomiasis. Methodology/Principal Findings A LAMP assay targeting a species specific sequence of S. haematobium ribosomal intergenic spacer was designed. The effectiveness of our LAMP was assessed in a number of patients´ urine samples with microscopy confirmed S. haematobium infection. For potentially large-scale application in field conditions, different DNA extraction methods, including a commercial kit, a modified NaOH extraction method and a rapid heating method were tested using small volumes of urine fractions (whole urine, supernatants and pellets). The heating of pellets from clinical samples was the most efficient method to obtain good-quality DNA detectable by LAMP. The detection limit of our LAMP was 1 fg/µL of S. haematobium DNA in urine samples. When testing all patients´ urine samples included in our study, diagnostic parameters for sensitivity and specificity were calculated for LAMP assay, 100% sensitivity (95% CI: 81.32%-100%) and 86.67% specificity (95% CI: 75.40%-94.05%), and also for microscopy detection of eggs in urine samples, 69.23% sensitivity (95% CI: 48.21% -85.63%) and 100% specificity (95% CI: 93.08%-100%). Conclusions/Significance We have developed and evaluated, for the first time, a LAMP assay for detection of S. haematobium DNA in heated pellets from patients´ urine samples using no complicated requirement procedure for DNA extraction. The procedure has been named the Rapid-Heat LAMPellet method and has the potential to be developed further as a field diagnostic tool for use in urogenital schistosomiasis-endemic areas. PMID:26230990

Polyethyleneimine-iron phosphate nanocomposite as a promising adsorbent for the isolation of DNA.

PubMed

Hu, Lin-Lin; Hu, Bo; Shen, Li-Ming; Zhang, Dan-Dan; Chen, Xu-Wei; Wang, Jian-Hua

2015-01-01

A polyethyleneimine (PEI)-iron phosphate (FePO4) nanocomposite is prepared by immobilization of PEI onto the surface of FePO4 nanoparticles via electrostatic interaction. The obtained PEI-FePO4 nanocomposites are spherical with a size centered in ca. 100 nm. They provide a novel adsorbent for the solid-phase extraction of DNA from complex sample matrices. At pH 4, 50 μg mL(-1) of DNA (salmon sperm DNA sodium salt) in 1.0 mL aqueous solution are quantitatively adsorbed (100%) by 2mg of the PEI-FePO4 nanocomposites, and meanwhile the coexisting albumin at a same concentration level is not retained, demonstrating the favorable selectivity of the nanocomposites to DNA against proteins. The adsorption behaviors of DNA onto the PEI-FePO4 nanocomposites fit Langmuir model, corresponding to an adsorption capacity of 61.88 mg g(-1). The adsorbed DNA could be readily recovered by using a 0.04 mol L(-1) Britton-Robinson (BR) buffer at pH 10, resulting in a recovery of 85%. The nanocomposites have been further used for the isolation of DNA from a series of real sample matrices, including synthetic λ-DNA sample, human whole blood and Escherichia coli cell lysate. The extraction efficiency and the purity of the recovered DNA are at least comparable to those achieved by using the reported sorbent materials or commercial kits. In addition, the DNAs isolated from human whole blood and E. coli cell lysate are of high quality, which have been further demonstrated by using them as templates for successful PCR amplifications. Copyright © 2014 Elsevier B.V. All rights reserved.

Anaplasma spp. in dogs and owners in north-western Morocco.

PubMed

Elhamiani Khatat, Sarah; Daminet, Sylvie; Kachani, Malika; Leutenegger, Christian M; Duchateau, Luc; El Amri, Hamid; Hing, Mony; Azrib, Rahma; Sahibi, Hamid

2017-04-24

Anaplasma phagocytophilum is an emerging tick-borne zoonotic pathogen of increased interest worldwide which has been detected in northern Africa. Anaplasma platys is also present in this region and could possibly have a zoonotic potential. However, only one recent article reports on the human esposure to A. phagocytophilum in Morocco and no data are available on canine exposure to both bacteria. Therefore, we conducted a cross-sectional epidemiological study aiming to assess both canine and human exposure to Anaplasma spp. in Morocco. A total of 425 dogs (95 urban, 160 rural and 175 working dogs) and 11 dog owners were sampled from four cities of Morocco. Canine blood samples were screened for Anaplasma spp. antibodies by an enzyme-linked immunosorbent assay (ELISA) and for A. phagocytophilum and A. platys DNA by a real-time polymerase chain reaction (RT-PCR) targeting the msp2 gene. Human sera were tested for specific A. phagocytophilum immunoglobulin G (IgG) using a commercial immunofluorescence assay (IFA) kit. Anaplasma spp. antibodies and A. platys DNA were detected in 21.9 and 7.5% of the dogs, respectively. Anaplasma phagocytophilum DNA was not amplified. Anaplasma platys DNA was significantly more frequently amplified for working dogs. No statistically significant differences in the prevalence of Anaplasma spp. antibodies or A. platys DNA detection were observed between sexes, age classes or in relation to exposure to ticks. A total of 348 Rhipicephalus sanguineus (sensu lato) ticks were removed from 35 urban and working dogs. The majority of dog owners (7/10) were seroreactive to A. phagoyctophilum IgG (one sample was excluded because of hemolysis). This study demonstrates the occurrence of Anaplasma spp. exposure and A. platys infection in dogs, and A. phagocytophilum exposure in humans in Morocco.

DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

PubMed

Dou, Baoting; Yang, Jianmei; Shi, Kai; Yuan, Ruo; Xiang, Yun

2016-09-15

We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of Quantifiler(®) Trio and InnoQuant™ human DNA quantification kits for detection of DNA degradation in developed and aged fingerprints.

PubMed

Goecker, Zachary C; Swiontek, Stephen E; Lakhtakia, Akhlesh; Roy, Reena

2016-06-01

The development techniques employed to visualize fingerprints collected from crime scenes as well as post-development ageing may result in the degradation of the DNA present in low quantities in such evidence samples. Amplification of the DNA samples with short tandem repeat (STR) amplification kits may result in partial DNA profiles. A comparative study of two commercially available quantification kits, Quantifiler(®) Trio and InnoQuant™, was performed on latent fingerprint samples that were either (i) developed using one of three different techniques and then aged in ambient conditions or (ii) undeveloped and then aged in ambient conditions. The three fingerprint development techniques used were: cyanoacrylate fuming, dusting with black powder, and the columnar-thin-film (CTF) technique. In order to determine the differences between the expected quantities and actual quantities of DNA, manually degraded samples generated by controlled exposure of DNA standards to ultraviolet radiation were also analyzed. A total of 144 fingerprint and 42 manually degraded DNA samples were processed in this study. The results indicate that the InnoQuant™ kit is capable of producing higher degradation ratios compared to the Quantifiler(®) Trio kit. This was an expected result since the degradation ratio is a relative value specific for a kit based on the length and extent of amplification of the two amplicons that vary from one kit to the other. Additionally, samples with lower concentrations of DNA yielded non-linear relationships of degradation ratio with the duration of aging, whereas samples with higher concentrations of DNA yielded quasi-linear relationships. None of the three development techniques produced a noticeably different degradation pattern when compared to undeveloped fingerprints, and therefore do not impede downstream DNA analysis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Human papilloma virus 18 detection in oral squamous cell carcinoma and potentially malignant lesions using saliva samples.

PubMed

Goot-Heah, Khor; Kwai-Lin, Thong; Froemming, Gabriele Ruth Anisah; Abraham, Mannil Thomas; Nik Mohd Rosdy, Nik Mohd Mazuan; Zain, Rosnah Binti

2012-01-01

Oral cancer has become one of the most prevalent cancers worldwide and human Papillomavirus is one of the risk factors for developing oral cancer. For this study HPV18 was chosen as it is one of the high risk HPV types and may lead to carcinogenesis. However, prevalence of HPV18 infection in Oral Squamous Cell Carcinoma in Malaysia remains unclear. This study aimed to investigate the viral load of HPV18 DNA in OSCC and potentially malignant lesions using saliva samples. Genomic DNAs of thirty saliva samples of normal subjects and thirty saliva samples compromised of 16 samples from potentially malignant lesions and 14 of OSCC patients were amplified for HPV18 DNA using a nested polymerase chain reaction analysis. All PCR products were then analyzed using the Bioanalyzer to confirm presence of HPV18 DNA. From thirty patients examined, only one of 30 (3.3%) cases was found to be positive for HPV18 in this study. The finding of this study revealed that there is a low viral detection of HPV18 in Malaysian OSCC by using saliva samples, suggesting that prevalence of HPV18 may not be important in this group of Malaysian OSCC.

Moisture content impacts the stability of DNA adsorbed onto gold microparticles.

PubMed

Smyth, Tyson J; Betker, Jamie; Wang, Wei; Anchordoquy, Thomas J

2011-11-01

Particle-mediated epidermal delivery (PMED) of small quantities of DNA (0.5-4.0 μg) has been reported to both induce an immune response and protect against disease in human subjects. In order for the PMED of DNA to be a viable technique for vaccination, the adsorbed DNA must be stable during shipping and storage. Here, we report that the storage stability of plasmid DNA adsorbed to 2-μm gold particles is strongly dependent on sample water content. Gold/DNA samples stored at 60°C and 6% relative humidity (RH) maintained supercoil content after 4-month storage, whereas storage at higher RHs facilitated degradation. Storage with desiccants had stabilizing effects on DNA similar to storage at 6% RH. However, storage with "indicating" Drierite and phosphorus pentoxide resulted in enhanced rates of DNA degradation. Copyright © 2011 Wiley-Liss, Inc.

Persistence of dead-cell bacterial DNA in ex vivo root canals and influence of nucleases on DNA decay in vitro.

PubMed

Brundin, Malin; Figdor, David; Roth, Chrissie; Davies, John K; Sundqvist, Göran; Sjögren, Ulf

2010-12-01

The fate of DNA from bacteria that do not survive in the root canal is uncertain, yet DNA longevity may confound recovery of authentic etiologic participants in the disease process. This study assessed the recovery of PCR-detectable DNA in ex vivo human root canals and some environmental factors on the decay of microbial DNA. Heat-killed Enterococcus faecalis cells were inoculated into instrumented human root canals ex vivo, and samples were taken at intervals over 2 years and analyzed by polymerase chain reaction. In an in vitro assay, heat-killed E. faecalis cells and extracted E. faecalis DNA were inoculated into various media, DNase, and culture of a DNase-producing species, Prevotella intermedia. Recovery of DNA was assessed by gel electrophoresis. In ex vivo human teeth, amplifiable DNA was recovered after 1 and 2 years (in 14/15 and 21/25 teeth, respectively). In vitro experiments showed that extracted DNA incubated in different media (water, 10%-50% sera, and DNase) progressively decomposed to levels below the detection limit. In corresponding assays, cell-bound DNA was more resistant to decay. Amplifiable DNA is preserved after cell death, but the critical determinant is the form of DNA. Free DNA undergoes spontaneous and enzymatic decomposition, whereas cell-bound E. faecalis DNA persists for long periods. Copyright © 2010 Mosby, Inc. All rights reserved.

The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing.

PubMed

Kennedy, Nicholas A; Walker, Alan W; Berry, Susan H; Duncan, Sylvia H; Farquarson, Freda M; Louis, Petra; Thomson, John M; Satsangi, Jack; Flint, Harry J; Parkhill, Julian; Lees, Charlie W; Hold, Georgina L

2014-01-01

Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/µL respectively (p<0.0001). Hierarchical clustering of sequence data by Yue and Clayton coefficient revealed four clusters. Samples from individuals H3 and I2 clustered by patient; however, samples from patient I1 extracted with the MoBio kit clustered with samples from patient H4 rather than the other I1 samples. Linear modelling on relative abundance of common bacterial families revealed significant differences between kits; samples extracted with MoBio Powersoil showed significantly increased Bacteroidaceae, Ruminococcaceae and Porphyromonadaceae, and lower Enterobacteriaceae, Lachnospiraceae, Clostridiaceae, and Erysipelotrichaceae (p<0.05). This study demonstrates significant differences in DNA yield and bacterial DNA composition when comparing DNA extracted from the same fecal sample with different extraction kits. This highlights the importance of ensuring that samples in a study are prepared with the same method, and the need for caution when cross-comparing studies that use different methods.

Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.

PubMed

Abbasi, Ibrahim; Kirstein, Oscar D; Hailu, Asrat; Warburg, Alon

2016-10-01

Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Meticulous plasma isolation is essential to avoid false low-level viraemia in Roche Cobas HIV-1 viral load assays.

PubMed

Mortier, Virginie; Vancoillie, Leen; Dauwe, Kenny; Staelens, Delfien; Demecheleer, Els; Schauvliege, Marlies; Dinakis, Sylvie; Van Maerken, Tom; Dessilly, Géraldine; Ruelle, Jean; Verhofstede, Chris

2017-10-24

Pre-analytical sample processing is often overlooked as a potential cause of inaccurate assay results. Here we demonstrate how plasma, extracted from standard EDTA-containing blood collection tubes, may contain traces of blood cells consequently resulting in a false low-level HIV-1 viral load when using Roche Cobas HIV-1 assays. The presence of human DNA in Roche Cobas 4800 RNA extracts and in RNA extracts from the Abbott HIV-1 RealTime assay was assessed by quantifying the human albumin gene by means of quantitative PCR. RNA was extracted from plasma samples before and after an additional centrifugation and tested for viral load and DNA contamination. The relation between total DNA content and viral load was defined. Elevated concentrations of genomic DNA were detected in 28 out of 100 Cobas 4800 extracts and were significantly more frequent in samples processed outside of the AIDS Reference Laboratory. An association between genomic DNA presence and spurious low-level viraemia results was demonstrated. Supplementary centrifugation of plasma before RNA extraction eliminated the contamination and the false viraemia. Plasma isolated from standard EDTA-containing blood collection tubes may contain traces of HIV DNA leading to false viral load results above the clinical cutoff. Supplementary centrifugation of plasma before viral load analysis may eliminate the occurrence of this spurious low-level viraemia.

Validation of a rapid DNA process with the RapidHIT® ID system using GlobalFiler® Express chemistry, a platform optimized for decentralized testing environments.

PubMed

Salceda, Susana; Barican, Arnaldo; Buscaino, Jacklyn; Goldman, Bruce; Klevenberg, Jim; Kuhn, Melissa; Lehto, Dennis; Lin, Frank; Nguyen, Phong; Park, Charles; Pearson, Francesca; Pittaro, Rick; Salodkar, Sayali; Schueren, Robert; Smith, Corey; Troup, Charles; Tsou, Dean; Vangbo, Mattias; Wunderle, Justus; King, David

2017-05-01

The RapidHIT ® ID is a fully automated sample-to-answer system for short tandem repeat (STR)-based human identification. The RapidHIT ID has been optimized for use in decentralized environments and processes presumed single source DNA samples, generating Combined DNA Index System (CODIS)-compatible DNA profiles in less than 90min. The system is easy to use, requiring less than one minute of hands-on time. Profiles are reviewed using centralized linking software, RapidLINK™ (IntegenX, Pleasanton, CA), a software tool designed to collate DNA profiles from single or multiple RapidHIT ID systems at different geographic locations. The RapidHIT ID has been designed to employ GlobalFiler ® Express and AmpFLSTR ® NGMSElect™, Thermo Fisher Scientific (Waltham, MA) STR chemistries. The Developmental Validation studies were performed using GlobalFiler ® Express with single source reference samples according to Scientific Working Group for DNA Analysis Methods guidelines. These results show that multiple RapidHIT ID systems networked with RapidLINK software form a highly reliable system for wide-scale deployment in locations such as police booking stations and border crossings enabling real-time testing of arrestees, potential human trafficking victims, and other instances where rapid turnaround is essential. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Molecular response of nasal mucosa to therapeutic exposure to broad-band ultraviolet radiation

PubMed Central

Mitchell, David; Paniker, Lakshmi; Sanchez, Guillermo; Bella, Zsolt; Garaczi, Edina; Szell, Marta; Hamid, Qutayba; Kemeny, Lajos; Koreck, Andrea

2010-01-01

Abstract Ultraviolet radiation (UVR) phototherapy is a promising new treatment for inflammatory airway diseases. However, the potential carcinogenic risks associated with this treatment are not well understood. UV-specific DNA photoproducts were used as biomarkers to address this issue. Radioimmunoassay was used to quantify cyclobutane pyrimidine dimers (CPDs) and (6–4) photoproducts in DNA purified from two milieus: nasal mucosa samples from subjects exposed to intranasal phototherapy and human airway (EpiAirway™) and human skin (EpiDerm™) tissue models. Immunohistochemistry was used to detect CPD formation and persistence in human nasal biopsies and human tissue models. In subjects exposed to broadband ultraviolet radiation, DNA damage frequencies were determined prior to as well as immediately after treatment and at increasing times post-treatment. We observed significant levels of DNA damage immediately after treatment and efficient removal of the damage within a few days. No residual damage was observed in human subjects exposed to multiple UVB treatments several weeks after the last treatment. To better understand the molecular response of the nasal epithelium to DNA damage, parallel experiments were conducted in EpiAirway and EpiDerm model systems. Repair rates in these two tissues were very similar and comparable to that observed in human skin. The data suggest that the UV-induced DNA damage response of respiratory epithelia is very similar to that of the human epidermis and that nasal mucosa is able to efficiently repair UVB induced DNA damage. PMID:18671762

Genome-wide DNA methylation levels and altered cortisol stress reactivity following childhood trauma in humans

PubMed Central

Houtepen, Lotte C.; Vinkers, Christiaan H.; Carrillo-Roa, Tania; Hiemstra, Marieke; van Lier, Pol A.; Meeus, Wim; Branje, Susan; Heim, Christine M.; Nemeroff, Charles B.; Mill, Jonathan; Schalkwyk, Leonard C.; Creyghton, Menno P.; Kahn, René S.; Joëls, Marian; Binder, Elisabeth B.; Boks, Marco P. M.

2016-01-01

DNA methylation likely plays a role in the regulation of human stress reactivity. Here we show that in a genome-wide analysis of blood DNA methylation in 85 healthy individuals, a locus in the Kit ligand gene (KITLG; cg27512205) showed the strongest association with cortisol stress reactivity (P=5.8 × 10−6). Replication was obtained in two independent samples using either blood (N=45, P=0.001) or buccal cells (N=255, P=0.004). KITLG methylation strongly mediates the relationship between childhood trauma and cortisol stress reactivity in the discovery sample (32% mediation). Its genomic location, a CpG island shore within an H3K27ac enhancer mark, and the correlation between methylation in the blood and prefrontal cortex provide further evidence that KITLG methylation is functionally relevant for the programming of stress reactivity in the human brain. Our results extend preclinical evidence for epigenetic regulation of stress reactivity to humans and provide leads to enhance our understanding of the neurobiological pathways underlying stress vulnerability. PMID:26997371

Does aflatoxin exposure in the United Kingdom constitute a cancer risk?

PubMed Central

Harrison, J C; Carvajal, M; Garner, R C

1993-01-01

Although the aflatoxins were discovered more than 30 years ago, there is still considerable controversy surrounding their human health effects. Most countries have introduced legislation to control the level of aflatoxins in food, but it is not known if these permitted levels still pose a significant cancer risk. Furthermore, it is unlikely that all the sources of human aflatoxin exposure have been discovered, nor if the liver is the only, or indeed, major target organ for aflatoxin-induced cancer in man. In our laboratory we have used both immunological and HPLC methods to examine human DNA from a variety of tissues and organs to identify and quantify aflatoxin DNA-adducts. We have already detected aflatoxin B1 (AFB1)-DNA adducts in formalin-fixed tissue from an acute poisoning incident in Southeast Asia. Here we have examined human colon and rectum DNA from normal and tumorous tissue obtained from cancer patients and colon, liver, pancreas, breast, and cervix DNA from autopsy specimens. AFB1-DNA adducts were detected in all tissue types examined and ranged from 0-60 adducts/10(6) nucleotides. Where sample size allowed, the adduct levels were confirmed by HPLC analysis. Tumor tissues tended to have higher adduct levels than normal tissue from the same individual, and levels generally increased with patient age. In samples analyzed by HPLC, the adducts present had the chromatographic properties of [8,9-dihydro-8-(N5-formyl)-2',5',6'-triamino-4'-oxo-(N5-pyramidyl) -9- hydroxy-aflatoxin B1, the ring-opened form of the AFB1-guanine adduct.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8319666

Quantitation of Human Papillomavirus DNA in Plasma of Oropharyngeal Carcinoma Patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cao Hongbin; Banh, Alice; Kwok, Shirley

Purpose: To determine whether human papillomavirus (HPV) DNA can be detected in the plasma of patients with HPV-positive oropharyngeal carcinoma (OPC) and to monitor its temporal change during radiotherapy. Methods and Materials: We used polymerase chain reaction to detect HPV DNA in the culture media of HPV-positive SCC90 and VU147T cells and the plasma of SCC90 and HeLa tumor-bearing mice, non-tumor-bearing controls, and those with HPV-negative tumors. We used real-time quantitative polymerase chain reaction to quantify the plasma HPV DNA in 40 HPV-positive OPC, 24 HPV-negative head-and-neck cancer patients and 10 non-cancer volunteers. The tumor HPV status was confirmed bymore » p16{sup INK4a} staining and HPV16/18 polymerase chain reaction or HPV in situ hybridization. A total of 14 patients had serial plasma samples for HPV DNA quantification during radiotherapy. Results: HPV DNA was detectable in the plasma samples of SCC90- and HeLa-bearing mice but not in the controls. It was detected in 65% of the pretreatment plasma samples from HPV-positive OPC patients using E6/7 quantitative polymerase chain reaction. None of the HPV-negative head-and-neck cancer patients or non-cancer controls had detectable HPV DNA. The pretreatment plasma HPV DNA copy number correlated significantly with the nodal metabolic tumor volume (assessed using {sup 18}F-deoxyglucose positron emission tomography). The serial measurements in 14 patients showed a rapid decline in HPV DNA that had become undetectable at radiotherapy completion. In 3 patients, the HPV DNA level had increased to a discernable level at metastasis. Conclusions: Xenograft studies indicated that plasma HPV DNA is released from HPV-positive tumors. Circulating HPV DNA was detectable in most HPV-positive OPC patients. Thus, plasma HPV DNA might be a valuable tool for identifying relapse.« less

Developmentally linked human DNA hypermethylation is associated with down-modulation, repression, and upregulation of transcription

PubMed Central

Baribault, Carl; Ehrlich, Kenneth C.; Ponnaluri, V. K. Chaithanya; Pradhan, Sriharsa; Lacey, Michelle; Ehrlich, Melanie

2018-01-01

ABSTRACT DNA methylation can affect tissue-specific gene transcription in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). To elucidate the variety of associations between differentiation-related DNA hypermethylation and transcription, we used available epigenomic and transcriptomic profiles from 38 human cell/tissue types to focus on such relationships in 94 genes linked to hypermethylated DMRs in myoblasts (Mb). For 19 of the genes, promoter-region hypermethylation in Mb (and often a few heterologous cell types) was associated with gene repression but, importantly, DNA hypermethylation was absent in many other repressed samples. In another 24 genes, DNA hypermethylation overlapped cryptic enhancers or super-enhancers and correlated with down-modulated, but not silenced, gene expression. However, such methylation was absent, surprisingly, in both non-expressing samples and highly expressing samples. This suggests that some genes need DMR hypermethylation to help repress cryptic enhancer chromatin only when they are actively transcribed. For another 11 genes, we found an association between intergenic hypermethylated DMRs and positive expression of the gene in Mb. DNA hypermethylation/transcription correlations similar to those of Mb were evident sometimes in diverse tissues, such as aorta and brain. Our findings have implications for the possible involvement of methylated DNA in Duchenne's muscular dystrophy, congenital heart malformations, and cancer. This epigenomic analysis suggests that DNA methylation is not simply the inevitable consequence of changes in gene expression but, instead, is often an active agent for fine-tuning transcription in association with development. PMID:29498561

A pilot study of gene expression analysis in workers with hand-arm vibration syndrome.

PubMed

Maeda, Setsuo; Yu, Xiaozhong; Wang, Rui-Sheng; Sakakibara, Hisataka

2008-04-01

The purpose of this pilot study was to examine differences in gene expressions by cDNA microarray analysis of hand-arm vibration syndrome (HAVS) patients. Vein blood samples were collected and total RNA was extracted. All blood samples were obtained in the morning in one visit after a standard light breakfast. We performed microarray analysis with the labeled cDNA prepared by reverse transcription from RNA samples, using the Human CHIP version 1 (DNA Chip Research Inc, Yokohama, Japan). There are 2,976 genes on the chip, and these genes were selected from a cDNA library prepared with human peripheral white blood cells (WBC). Different gene levels between the HAVS patients and controls, and between groups of HAVS with different levels of symptoms, were indicated by the randomized variance model. The most up-regulated genes were analyzed for their possible functions and association with the occurrence of HAVS. From the results of this pilot study, although the results were obtained a limited number of subjects, it would appear that cDNA microarray analysis of HAVS patients has potential as a new objective method of HAVS diagnosis. Further research is needed to examine the gene expression with increased numbers of patients at different stages of HAVS.

Human HST1 (HSTF1) gene maps to chromosome band 11q13 and coamplifies with the INT2 gene in human cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshida, Michihiro C.; Wada, Makio; Satoh, Hitoshi

1988-07-01

The human HST1 gene, previously designated the hst gene, and now assigned the name HSTF1 for heparin-binding secretory transforming factor in human gene nomenclature, was originally identified as a transforming gene in DNAs from human stomach cancers by transfection assay with mouse NIH 3T3 cells. The amino acid sequence of the product deduced from DNA sequences of the HST1 cDNA and genomic clones had approximately 40% homology to human basic and acidic fibroblast growth factors and mouse Int-2-encoded protein. The authors have mapped the human HST1 gene to chromosome 11 at band q13.3 by Southern blot hybridization analysis of amore » panel of human and mouse somatic cell hybrids and in situ hybridization with an HST1 cDNA probe. The HST1 gene was found to be amplified in DNAs obtained from a stomach cancer and a vulvar carcinoma cell line, A431. In all of these samples of DNA, the INT2 gene, previously mapped to human chromosome 11q13, was also amplified to the same degree as the HST1 gene.« less

Comparison of the Quantiplex Version 3.0 Assay and a Sensitized Amplicor Monitor Assay for Measurement of Human Immunodeficiency Virus Type 1 RNA Levels in Plasma Samples

PubMed Central

Highbarger, Helene C.; Alvord, W. Gregory; Jiang, Min Kang; Shah, Akram S.; Metcalf, Julia A.; Lane, H. Clifford; Dewar, Robin L.

1999-01-01

This study evaluated correlation and agreement between version 3 of the Quantiplex human immunodeficiency virus type 1 (HIV-1) RNA assay (v3 branched DNA [bDNA]) and a sensitized Amplicor HIV-1 Monitor assay (reverse transcription [RT]-PCR) for the measurement of HIV RNA. Three hundred eighteen samples from 59 randomly selected, HIV-1-seropositive persons on various drug protocols from the National Institute of Allergy and Infectious Diseases HIV outpatient clinic were studied. The results indicate that v3 bDNA and RT-PCR are highly correlated (r = 0.98) and are in good agreement (mean difference in log10 copies/ml ± 2 standard deviations = 0.072 ± 0.371). The relationship between values obtained by both assays is given by the following equation: log10v3 bDNA = −0.0915 + 1.0052 · log10RT-PCR. This represents a 1.026-fold difference between log10RT-PCR values and log10v3 bDNA values. PMID:10523562

High-resolution melting PCR assay, applicable for diagnostics and screening studies, allowing detection and differentiation of several Babesia spp. infecting humans and animals.

PubMed

Rozej-Bielicka, Wioletta; Masny, Aleksander; Golab, Elzbieta

2017-10-01

The goal of the study was to design a single tube PCR test for detection and differentiation of Babesia species in DNA samples obtained from diverse biological materials. A multiplex, single tube PCR test was designed for amplification of approximately 400 bp region of the Babesia 18S rRNA gene. Universal primers were designed to match DNA of multiple Babesia spp. and to have low levels of similarity to DNA sequences of other intracellular protozoa and Babesia hosts. The PCR products amplified from Babesia DNA isolated from human, dog, rodent, deer, and tick samples were subjected to high-resolution melting analysis for Babesia species identification. The designed test allowed detection and differentiation of four Babesia species, three zoonotic (B. microti, B. divergens, B. venatorum) and one that is generally not considered zoonotic-Babesia canis. Both detection and identification of all four species were possible based on the HRM curves of the PCR products in samples obtained from the following: humans, dogs, rodents, and ticks. No cross-reactivity with DNA of Babesia hosts or Plasmodium falciparum and Toxoplasma gondii was observed. The lack of cross-reactivity with P. falciparum DNA might allow using the assay in endemic malaria areas. The designed assay is the first PCR-based test for detection and differentiation of several Babesia spp. of medical and veterinary importance, in a single tube reaction. The results of the study show that the designed assay for Babesia detection and identification could be a practical and inexpensive tool for diagnostics and screening studies of diverse biological materials.

Perinatal transmission of human papilomavirus DNA

PubMed Central

Rombaldi, Renato L; Serafini, Eduardo P; Mandelli, Jovana; Zimmermann, Edineia; Losquiavo, Kamille P

2009-01-01

The purpose was to study the perinatal transmission of human papillomavirus DNA (HPV-DNA) in 63 mother-newborn pairs, besides looking at the epidemiological factors involved in the viral DNA transmission. The following sampling methods were used: (1) in the pregnant woman, when was recruited, in cervix and clinical lesions of the vagina, vulva and perineal region; (2) in the newborn, (a) buccal, axillary and inguinal regions; (b) nasopharyngeal aspirate, and (c) cord blood; (3) in the children, buccal was repeated in the 4th week and 6th and 12th month of life. HPV-DNA was identified using two methodologies: multiplex PCR (PGMY09 and MY11 primers) and nested-PCR (genotypes 6/11, 16, 18, 31, 33, 42, 52 and 58). Perinatal transmission was considered when concordance was found in type-specific HPV between mother/newborn or mother/child. HPV-DNA genital was detected in 49 pregnant women submitted to delivery. Eleven newborns (22.4%, n = 11/49) were HPV-DNA positive. In 8 cases (16.3%, n = 8/49) there was type specific HPV concordance between mother/newborn samples. At the end of the first month of life three children (6.1%, n = 3/49) became HPV-DNA positive, while two remained positive from birth. In 3 cases (100%, n = 3/3) there was type specific HPV concordance between mother/newborn samples. In the 6th month, a child (2%, n = 1/49) had become HPV-DNA positive between the 1st and 6th month of life, and there was type specific HPV concordance of mother/newborn samples. All the HPV-DNA positive children (22.4%, n = 11/49) at birth and at the end first month of life (6.1%, n = 3/49) became HPV-DNA negative at the age of 6 months. The HPV-DNA positive child (2%, n = 1/49) from 1st to the 6th month of life became HPV-DNA negative between the 6th and 12th month of life and one child had anogenital warts. In the twelfth month all (100%, n = 49/49) the children studied were HPV-DNA negative. A positive and significant correlation was observed between perinatal transmission of HPV-DNA and the immunodepression of maternal variables (HIV, p = 0.007). Finally, the study suggests that perinatal transmission of HPV-DNA occurred in 24.5% (n = 12/49) of the cases studied. PMID:19545396

Tamoxifen-DNA adduct formation in monkey and human reproductive organs.

PubMed

Hernandez-Ramon, Elena E; Sandoval, Nicole A; John, Kaarthik; Cline, J Mark; Wood, Charles E; Woodward, Ruth A; Poirier, Miriam C

2014-05-01

The estrogen analog tamoxifen (TAM), used for adjuvant therapy of breast cancer, induces endometrial and uterine tumors in breast cancer patients. Proliferation stimulus of the uterine endometrium is likely involved in tumor induction, but genotoxicity may also play a role. Formation of TAM-DNA adducts in human tissues has been reported but remains controversial. To address this issue, we examined TAM-DNA adducts in uteri from two species of monkeys, Erythrocebus patas (patas) and Macaca fascicularis (macaque), and in human endometrium and myometrium. Monkeys were given 3-4 months of chronic TAM dosing scaled to be equivalent to the daily human dose. In the uteri, livers and brains from the patas (n = 3), and endometrium from the macaques (n = 4), TAM-DNA adducts were measurable by TAM-DNA chemiluminescence immunoassay. Average TAM-DNA adduct values for the patas uteri (23 adducts/10(8) nucleotides) were similar to those found in endometrium of the macaques (19 adducts/10(8) nucleotides). Endometrium of macaques exposed to both TAM and low-dose estradiol (n = 5) averaged 34 adducts/10(8) nucleotides. To examine TAM-DNA persistence in the patas, females (n = 3) were exposed to TAM for 3 months and to no drug for an additional month, resulting in low or non-detectable TAM-DNA in livers and uteri. Human endometrial and myometrial samples from women receiving (n = 8) and not receiving (n = 8) TAM therapy were also evaluated. Women receiving TAM therapy averaged 10.3 TAM-DNA adducts/10(8) nucleotides, whereas unexposed women showed no detectable TAM-DNA. The data indicate that genotoxicity, in addition to estrogen agonist effects, may contribute to TAM-induced human endometrial cancer.

Evidence of SV40 infections in hospitalized children

NASA Technical Reports Server (NTRS)

Butel, J. S.; Jafar, S.; Wong, C.; Arrington, A. S.; Opekun, A. R.; Finegold, M. J.; Adam, E.

1999-01-01

Simian virus 40 (SV40) is known to have contaminated poliovirus vaccines used between 1955 and 1963. Accumulating reports have described the presence of SV40 DNA in human tumors and normal tissues, although the significance of human infections by SV40 is unknown. We investigated whether unselected hospitalized children had evidence of SV40 infections and whether any clinical correlations were apparent. Serum samples were examined for SV40 neutralizing antibody using a specific plaque reduction test; of 337 samples tested, 20 (5.9%) had antibody to SV40. Seropositivity increased with age and was significantly associated with kidney transplants (6 of 15 [40%] positive, P < .001). Many of the antibody-positive patients had impaired immune systems. Molecular assays (polymerase chain reaction and DNA sequence analysis) on archival tissue specimens confirmed the presence of SV40 DNA in 4 of the antibody-positive patients. This study, using 2 independent assays, shows the presence of SV40 infections in children born after 1980. We conclude that SV40 causes natural infections in humans.

Screening of Viral Pathogens from Pediatric Ileal Tissue Samples after Vaccination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hewitson, Laura; Thissen, James B.; Gardner, Shea N.

In 2010, researchers reported that the two US-licensed rotavirus vaccines contained DNA or DNA fragments from porcine circovirus (PCV). Although PCV, a common virus among pigs, is not thought to cause illness in humans, these findings raised several safety concerns. In this study, we sought to determine whether viruses, including PCV, could be detected in ileal tissue samples of children vaccinated with one of the two rotavirus vaccines. A broad spectrum, novel DNA detection technology, the Lawrence Livermore Microbial Detection Array (LLMDA), was utilized, and confirmation of viral pathogens using the polymerase chain reaction (PCR) was conducted. The LLMDA technologymore » was recently used to identify PCV from one rotavirus vaccine. Ileal tissue samples were analyzed from 21 subjects, aged 15–62 months. PCV was not detected in any ileal tissue samples by the LLMDA or PCR. LLMDA identified a human rotavirus A from one of the vaccinated subjects, which is likely due to a recent infection from a wild type rotavirus. LLMDA also identified human parechovirus, a common gastroenteritis viral infection, from two subjects. Additionally, LLMDA detected common gastrointestinal bacterial organisms from the Enterobacteriaceae , Bacteroidaceae , and Streptococcaceae families from several subjects. This study provides a survey of viral and bacterial pathogens from pediatric ileal samples, and may shed light on future studies to identify pathogen associations with pediatric vaccinations.« less

Screening of Viral Pathogens from Pediatric Ileal Tissue Samples after Vaccination

DOE PAGES

Hewitson, Laura; Thissen, James B.; Gardner, Shea N.; ...

2014-01-01

In 2010, researchers reported that the two US-licensed rotavirus vaccines contained DNA or DNA fragments from porcine circovirus (PCV). Although PCV, a common virus among pigs, is not thought to cause illness in humans, these findings raised several safety concerns. In this study, we sought to determine whether viruses, including PCV, could be detected in ileal tissue samples of children vaccinated with one of the two rotavirus vaccines. A broad spectrum, novel DNA detection technology, the Lawrence Livermore Microbial Detection Array (LLMDA), was utilized, and confirmation of viral pathogens using the polymerase chain reaction (PCR) was conducted. The LLMDA technologymore » was recently used to identify PCV from one rotavirus vaccine. Ileal tissue samples were analyzed from 21 subjects, aged 15–62 months. PCV was not detected in any ileal tissue samples by the LLMDA or PCR. LLMDA identified a human rotavirus A from one of the vaccinated subjects, which is likely due to a recent infection from a wild type rotavirus. LLMDA also identified human parechovirus, a common gastroenteritis viral infection, from two subjects. Additionally, LLMDA detected common gastrointestinal bacterial organisms from the Enterobacteriaceae , Bacteroidaceae , and Streptococcaceae families from several subjects. This study provides a survey of viral and bacterial pathogens from pediatric ileal samples, and may shed light on future studies to identify pathogen associations with pediatric vaccinations.« less

[Genetic profiling of Giardia intestinalis by polimerase chain in human and dogs samples of Colombian Caribean Coast].

PubMed

Arroyo-Salgado, Bárbara; Buelvas-Montes, Yaleyvis; Villalba-Vizcaíno, Vivian; Salomón-Arzuza, Octavio

2014-01-01

Giardia intestinalis (G. Intestinalis) is a protozoan that causes diarrheal disease and malabsorption syndrome in humans and other mammals. It presents a high genetic diversity evidenced in the recognition of 7 genotypes (A-G). Genotypes A and B are commonly associated to humans and domestic animals such as dogs. The aim of this study was to conduct a preliminary genetic characterization of G. intestinalis in humans and dogs from two cities on the Caribbean coast of Colombia. Sampling areas were selected according to the highest numbers of acute diarrheal disease. Stool samples were collected from children under 7 years old, with positive medical tests for G. intestinalis. Cysts were purified by sucrose gradient and DNA samples were isolated by extraction with organic solvents. Molecular characterization was performed by amplifying the gene triose phosphate isomerase (tpi) by using a semi-nested PCR. A total of 202 samples of DNA were obtained; of these, 111 were positive in coproparasitological analysis (13 dogs and 98 children). Genotype distribution in positive samples was: 5.1% belonged to genotype A and 92.3% to genotype B. Genotype B was present in humans and animals. The most common genotype in both human and animal samples was genotype B, suggesting a zoonotic transmission cycle. Copyright © 2012 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Association of human herpesvirus 6 subtypes with symptomatic apical periodontitis.

PubMed

Hernádi, Katinka; Csoma, Eszter; Adám, Balázs; Szalmás, Anita; Gyöngyösi, Eszter; Veress, György; Ildikó-Márton; Kónya, József

2011-09-01

The occurrence of human herpesvirus (HHV) 6 subtypes A and B in apical periodontitis was determined. The relationship of HHV-6 subtypes to other disease associated herpesviruses, i.e., Epstein-Barr virus (EBV) and human cytomegalovirus, was also investigated. Forty apical periodontitis samples (17 symptomatic and 23 asymptomatic) and 40 healthy pulp control samples were collected. Nested polymerase chain reaction was used to detect HHV-6 DNA. HHV-6 DNA was observed in significantly higher frequencies in apical periodontitis samples than in control samples (20% vs. 2.5%; P = .03). Further classification of apical lesions revealed that subtype B of HHV-6 was significantly associated with large-sized and symptomatic lesions (P < .01). Thirty-one apical lesions (77%) harbored ≥1 of the tested herpesviruses: EBV was the most frequent herpesvirus (72.5%) in apical periodontitis, followed by HHV-6 (20%). Our findings suggest that EBV and HHV-6B infections can be associated with symptomatic apical periodontitis. Copyright © 2011 Mosby, Inc. All rights reserved.

Molecular detection of Borrelia bissettii DNA in serum samples from patients in the Czech Republic with suspected borreliosis.

PubMed

Rudenko, Nataliia; Golovchenko, Maryna; Růzek, Daniel; Piskunova, Natalja; Mallátová, Nadja; Grubhoffer, Libor

2009-03-01

Until recently, three spirochete genospecies were considered to be the causative agents of Lyme borreliosis (LB) in Europe: Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. However, the DNA of Borrelia valaisiana, Borrelia lusitaniae, Borrelia spielmanii and Borrelia bissettii has already been detected in samples of human origin, or the spirochetes were isolated from the patients with symptoms of LB. Molecular analysis of 12 selected serum samples collected in the regional hospital confirmed the presence of B. bissettii DNA in cases of single and multiple infection in patients with symptomatic borreliosis or chronic borrelial infection. The presence of B. bissettii as a single strain in patients provides strong support of the fact that B. bissettii might be a causative agent of the disease. After the first isolation of B. bissettii from the samples of human origin in Slovenia, following the detection of this species in cardiac valve tissue of the patient with endocarditis and aortic valve stenosis in the Czech Republic, here we present additional molecular data supporting the involvement of B. bissettii in LB in Europe.

Y Chromosome DNA in Women's Vaginal Samples as a Biomarker of Recent Vaginal Sex and Condom Use With Male Partners in the HPV Infection and Transmission Among Couples Through Heterosexual Activity Cohort Study.

PubMed

Malagón, Talía; Burchell, Ann; El-Zein, Mariam; Guénoun, Julie; Tellier, Pierre-Paul; Coutlée, François; Franco, Eduardo L

2018-01-01

Y chromosome DNA from male epithelial and sperm cells was detected in vaginal samples after unprotected sex in experimental studies. We assessed the strength of this association in an observational setting to examine the utility of Y chromosome DNA as a biomarker of recent sexual behaviors in epidemiological studies. The HPV (human papillomavirus) Infection and Transmission Among Couples Through Heterosexual Activity cohort study enrolled 502 women attending a university or college in Montréal, Canada, and their male partners from 2005 to 2010. Participants completed self-administered questionnaires. We used real-time polymerase chain reaction to test women's baseline vaginal samples for Y chromosome DNA and assessed which sexual behaviors were independent predictors of Y chromosome DNA positivity and quantity with logistic and negative binomial regression. Y chromosome DNA positivity decreased from 77% in women in partnerships reporting vaginal sex 0 to 1 day ago to 13% in women in partnerships reporting last vaginal sex of 15 or more days ago (adjusted odds ratio, 0.09; 95% confidence interval, 0.02-0.36). The mean proportion of exfoliated vaginal sample cells with Y chromosome DNA was much lower for women who reported always using condoms (0.01%) than for women who reported never using condoms (2.07%) (adjusted ratio, 26.8; 95% confidence interval, 8.9-80.5). No association was found with reported oral/digital sex frequency or concurrency of partnerships. Y chromosome DNA quantity is strongly associated with days since last vaginal sex and lack of condom use in observational settings. Y chromosome DNA quantity may prove useful as a correlate of recent vaginal sex in observational studies lacking data on sexual behavior, such as surveillance studies of human papillomavirus infection prevalence.

Waterborne Viruses and F-Specific Coliphages in Mixed-Use Watersheds: Microbial Associations, Host Specificities, and Affinities with Environmental/Land Use Factors

PubMed Central

Jones, Tineke H.; Brassard, Julie; Topp, Edward; Wilkes, Graham

2016-01-01

ABSTRACT From the years 2008 to 2014, a total of 1,155 water samples were collected (spring to fall) from 24 surface water sampling sites located in a mixed-used but predominantly agricultural (i.e., dairy livestock production) river basin in eastern Ontario, Canada. Water was analyzed for viable F-specific DNA (F-DNA) and F-specific RNA (F-RNA) (genogroup I [GI] to GIV) coliphage and a suite of molecularly detected viruses (norovirus [GI to GIV], torque teno virus [TTV], rotavirus, kobuvirus, adenovirus, astrovirus, hepatitis A, and hepatitis E). F-DNA and F-RNA coliphage were detected in 33 and 28% of the samples at maximum concentrations of 2,000 and 16,300 PFU · 100 ml−1, respectively. Animal TTV, human TTV, kobuvirus, astrovirus, and norovirus GIII were the most prevalent viruses, found in 23, 20, 13, 12, and 11% of samples, respectively. Viable F-DNA coliphage was found to be a modest positive indicator of molecularly detected TTV. F-RNA coliphage, unlike F-DNA coliphage, was a modest positive predictor of norovirus and rotavirus. There were, however, a number of significant negative associations among F-specific coliphage and viruses. F-DNA coliphage densities of >142 PFU · 100 ml−1 delineated conditions when ∼95% of water samples contained some type of virus. Kobuvirus was the virus most strongly related to detection of any other virus. Land use had some associations with virus/F-specific coliphage detection, but season and surface water flow were the variables that were most important for broadly delineating detection. Higher relative levels of detection of human viruses and human F-RNA coliphage were associated with higher relative degrees of upstream human land development in a catchment. IMPORTANCE This study is one of the first, to our knowledge, to evaluate relationships among F-specific coliphages and a large suite of enteric viruses in mixed-use but agriculturally dominated surface waters in Canada. This study suggested that relationships between viable F-specific coliphages and molecularly detected viruses do exist, but they are not always positive. Caution should be employed if viable F-specific coliphages are to be used as indicators of virus presence in surface waters. This study elucidates relative effects of agriculture, wildlife, and human activity on virus and F-specific coliphage detection. Seasonal and meteorological attributes play a strong role in the detection of most virus and F-specific coliphage targets. PMID:27836843

Waterborne Viruses and F-Specific Coliphages in Mixed-Use Watersheds: Microbial Associations, Host Specificities, and Affinities with Environmental/Land Use Factors.

PubMed

Jones, Tineke H; Brassard, Julie; Topp, Edward; Wilkes, Graham; Lapen, David R

2017-02-01

From the years 2008 to 2014, a total of 1,155 water samples were collected (spring to fall) from 24 surface water sampling sites located in a mixed-used but predominantly agricultural (i.e., dairy livestock production) river basin in eastern Ontario, Canada. Water was analyzed for viable F-specific DNA (F-DNA) and F-specific RNA (F-RNA) (genogroup I [GI] to GIV) coliphage and a suite of molecularly detected viruses (norovirus [GI to GIV], torque teno virus [TTV], rotavirus, kobuvirus, adenovirus, astrovirus, hepatitis A, and hepatitis E). F-DNA and F-RNA coliphage were detected in 33 and 28% of the samples at maximum concentrations of 2,000 and 16,300 PFU · 100 ml -1 , respectively. Animal TTV, human TTV, kobuvirus, astrovirus, and norovirus GIII were the most prevalent viruses, found in 23, 20, 13, 12, and 11% of samples, respectively. Viable F-DNA coliphage was found to be a modest positive indicator of molecularly detected TTV. F-RNA coliphage, unlike F-DNA coliphage, was a modest positive predictor of norovirus and rotavirus. There were, however, a number of significant negative associations among F-specific coliphage and viruses. F-DNA coliphage densities of >142 PFU · 100 ml -1 delineated conditions when ∼95% of water samples contained some type of virus. Kobuvirus was the virus most strongly related to detection of any other virus. Land use had some associations with virus/F-specific coliphage detection, but season and surface water flow were the variables that were most important for broadly delineating detection. Higher relative levels of detection of human viruses and human F-RNA coliphage were associated with higher relative degrees of upstream human land development in a catchment. This study is one of the first, to our knowledge, to evaluate relationships among F-specific coliphages and a large suite of enteric viruses in mixed-use but agriculturally dominated surface waters in Canada. This study suggested that relationships between viable F-specific coliphages and molecularly detected viruses do exist, but they are not always positive. Caution should be employed if viable F-specific coliphages are to be used as indicators of virus presence in surface waters. This study elucidates relative effects of agriculture, wildlife, and human activity on virus and F-specific coliphage detection. Seasonal and meteorological attributes play a strong role in the detection of most virus and F-specific coliphage targets. © Crown copyright 2017.

Assessment of MagNA pure LC extraction system for detection of human papillomavirus (HPV) DNA in PreservCyt samples by the Roche AMPLICOR and LINEAR ARRAY HPV tests.

PubMed

Stevens, Matthew P; Rudland, Elice; Garland, Suzanne M; Tabrizi, Sepehr N

2006-07-01

Roche Molecular Systems recently released two PCR-based assays, AMPLICOR and LINEAR ARRAY (LA), for the detection and genotyping, respectively, of human papillomaviruses (HPVs). The manual specimen processing method recommended for use with both assays, AmpliLute, can be time-consuming and labor-intensive and is open to potential specimen cross-contamination. We evaluated the Roche MagNA Pure LC (MP) as an alternative for specimen processing prior to use with either assay. DNA was extracted from cervical brushings, collected in PreservCyt media, by AmpliLute and MP using DNA-I and Total Nucleic Acid (TNA) kits, from 150 patients with histologically confirmed cervical abnormalities. DNA was amplified and detected by AMPLICOR and the LA HPV test. Concordances of 96.5% (139 of 144) (kappa=0.93) and 95.1% (135 of 142) (kappa=0.90) were generated by AMPLICOR when we compared DNA extracts from AmpliLute to MP DNA-I and TNA, respectively. The HPV genotype profiles were identical in 78.7 and 74.7% of samples between AmpliLute and DNA-I or TNA, respectively. To improve LA concordance, all 150 specimens were extracted by MP DNA-I protocol after the centrifugation of 1-ml PreservCyt samples. This modified approach improved HPV genotype concordance levels between AmpliLute and MP DNA-I to 88.0% (P=0.043) without affecting AMPLICOR sensitivity. Laboratories that have an automated MP extraction system would find this procedure more feasible and easier to handle than the recommended manual extraction method and could substitute such extractions for AMPLICOR and LA HPV tests once internally validated.

Searching for Helicobacter pylori and Chlamydia pneumoniae in primary endodontic infections.

PubMed

Rôças, Isabela N; Siqueira, José F

2012-04-01

The purpose of this study was to search samples from primary endodontic infections for the presence of two common human bacterial pathogens - Helicobacter pylori and Chlamydia pneumoniae. Genomic DNA isolated from samples taken from 25 root canals of teeth with asymptomatic (chronic) apical periodontitis and 25 aspirates from acute apical abscess was initially amplified by the multiple displacement amplification approach and then used as template in species-specific polymerase chain reaction (PCR) for detection of H. pylori and C. pneumoniae. All clinical samples were positive for the presence of bacterial DNA. However, no clinical sample was positive for either H. pylori or C. pneumoniae. Neither H. pylori nor C. pneumoniae were found in samples from primary endodontic infections. These findings suggest that these species are not candidate endodontic pathogens and that the necrotic root canal does not serve as a reservoir for these human pathogens in healthy patients.

Rectal swab screening assays of public health importance in molecular diagnostics: Sample adequacy control.

PubMed

Glisovic, Sanja; Eintracht, Shaun; Longtin, Yves; Oughton, Matthew; Brukner, Ivan

Rectal swabs are routinely used by public health authorities to screen for multi-drug resistant enteric bacteria including vancomycin-resistant enterococci (VRE) and carbapenem-resistant enterobacteriaceae (CRE). Screening sensitivity can be influenced by the quality of the swabbing, whether performed by the patient (self-swabbing) or a healthcare practitioner. One common exclusion criterion for rectal swabs is absence of "visible soiling" from fecal matter. In our institution, this criterion excludes almost 10% of rectal swabs received in the microbiology laboratory. Furthermore, over 30% of patients in whom rectal swabs are cancelled will not be re-screened within the next 48h, resulting in delays in removing infection prevention measures. We describe two quantitative polymerase chain reaction (qPCR)-based assays, human RNAse P and eubacterial 16S rDNA, which might serve as suitable controls for sampling adequacy. However, lower amounts of amplifiable human DNA make the 16s rDNA assay a better candidate for sample adequacy control. Copyright © 2017. Published by Elsevier Ltd.

No detection of SV40 DNA in mesothelioma tissues from a high incidence area in Sweden.

PubMed

Lundstig, Annika; Dejmek, Annika; Eklund, Carina; Filinic, Ivo; Dillner, Joakim

2007-01-01

Simian virus 40 (SV40), a polyoma virus of the rhesus macaque was discovered in 1960 as a contaminant of human polio vaccines produced in monkey cells. A number of studies have reported the detection of SV40 nucleotide sequences in human tumors, mainly mesotheliomas, but the reports have not been consistent. The presence of SV40 in 26 consecutive cases of malignant mesothelioma of biphasic type was investigated using a SV40 quantitative real time polymerase chain reaction (PCR) with a sensitivity of 10 copies of viral DNA per sample. All the samples were also tested for amplifiability using a real-time PCR for the beta-globin gene. Eighteen tumors were amplifiable, but none contained SV40 DNA. The results do not support an association between mesothelioma and SV40.

Water as Source of Francisella tularensis Infection in Humans, Turkey

PubMed Central

Kilic, Selcuk; Birdsell, Dawn N.; Karagöz, Alper; Çelebi, Bekir; Bakkaloglu, Zekiye; Arikan, Muzaffer; Sahl, Jason W.; Mitchell, Cedar; Rivera, Andrew; Maltinsky, Sara; Keim, Paul; Üstek, Duran; Durmaz, Rıza

2015-01-01

Francisella tularensis DNA extractions and isolates from the environment and humans were genetically characterized to elucidate environmental sources that cause human tularemia in Turkey. Extensive genetic diversity consistent with genotypes from human outbreaks was identified in environmental samples and confirmed water as a source of human tularemia in Turkey. PMID:26583383

Vitrification of neat semen alters sperm parameters and DNA integrity.

PubMed

Khalili, Mohammad Ali; Adib, Maryam; Halvaei, Iman; Nabi, Ali

2014-05-06

Our aim was to evaluate the effect of neat semen vitrification on human sperm vital parameters and DNA integrity in men with normal and abnormal sperm parameters. Semen samples were 17 normozoospermic samples and 17 specimens with abnormal sperm parameters. Semen analysis was performed according to World Health Organization (WHO) criteria. Then, the smear was provided from each sample and fixed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Vitrification of neat semen was done by plunging cryoloops directly into liquid nitrogen and preserved for 7 days. The samples were warmed and re-evaluated for sperm parameters as well as DNA integrity. Besides, the correlation between sperm parameters and DNA fragmentation was assessed pre- and post vitrification. Cryopreserved spermatozoa showed significant decrease in sperm motility, viability and normal morphology after thawing in both normal and abnormal semen. Also, the rate of sperm DNA fragmentation was significantly higher after vitrification compared to fresh samples in normal (24.76 ± 5.03 and 16.41 ± 4.53, P = .002) and abnormal (34.29 ± 10.02 and 23.5 ± 8.31, P < .0001), respectively. There was negative correlation between sperm motility and sperm DNA integrity in both groups after vitrification. Vitrification of neat ejaculates has negative impact on sperm parameters as well as DNA integrity, particularly among abnormal semen subjects. It is, therefore, recommend to process semen samples and vitrify the sperm pellets.

DNA preservation in skeletal elements from the World Trade Center disaster: recommendations for mass fatality management.

PubMed

Mundorff, Amy Z; Bartelink, Eric J; Mar-Cash, Elaine

2009-07-01

The World Trade Center (WTC) victim identification effort highlights taphonomic influences on the degradation of DNA from victims of mass fatality incidents. This study uses a subset of the WTC-Human Remains Database to evaluate differential preservation of DNA by skeletal element. Recovery location, sex, and victim type (civilian, firefighter, or plane passenger) do not appear to influence DNA preservation. Results indicate that more intact elements, as well as elements encased in soft tissue, produced slightly higher identification rates than more fragmented remains. DNA identification rates by element type conform to previous findings, with higher rates generally found in denser, weight-bearing bones. However, smaller bones including patellae, metatarsals, and foot phalanges yielded rates comparable to both femora and tibiae. These elements can be easily sampled with a disposable scalpel, and thus reduce potential DNA contamination. These findings have implications for DNA sampling guidelines in future mass fatality incidents.

Comparison of five bacteriophages as models for viral aerosol studies.

PubMed

Turgeon, Nathalie; Toulouse, Marie-Josée; Martel, Bruno; Moineau, Sylvain; Duchaine, Caroline

2014-07-01

Bacteriophages are perceived to be good models for the study of airborne viruses because they are safe to use, some of them display structural features similar to those of human and animal viruses, and they are relatively easy to produce in large quantities. Yet, only a few studies have investigated them as models. It has previously been demonstrated that aerosolization, environmental conditions, and sampling conditions affect viral infectivity, but viral infectivity is virus dependent. Thus, several virus models are likely needed to study their general behavior in aerosols. The aim of this study was to compare the effects of aerosolization and sampling on the infectivity of five tail-less bacteriophages and two pathogenic viruses: MS2 (a single-stranded RNA [ssRNA] phage of the Leviviridae family), Φ6 (a segmented double-stranded RNA [dsRNA] phage of the Cystoviridae family), ΦX174 (a single-stranded DNA [ssDNA] phage of the Microviridae family), PM2 (a double-stranded DNA [dsDNA] phage of the Corticoviridae family), PR772 (a dsDNA phage of the Tectiviridae family), human influenza A virus H1N1 (an ssRNA virus of the Orthomyxoviridae family), and the poultry virus Newcastle disease virus (NDV; an ssRNA virus of the Paramyxoviridae family). Three nebulizers and two nebulization salt buffers (with or without organic fluid) were tested, as were two aerosol sampling devices, a liquid cyclone (SKC BioSampler) and a dry cyclone (National Institute for Occupational Safety and Health two-stage cyclone bioaerosol sampler). The presence of viruses in collected air samples was detected by culture and quantitative PCR (qPCR). Our results showed that these selected five phages behave differently when aerosolized and sampled. RNA phage MS2 and ssDNA phage ΦX174 were the most resistant to aerosolization and sampling. The presence of organic fluid in the nebulization buffer protected phages PR772 and Φ6 throughout the aerosolization and sampling with dry cyclones. In this experimental setup, the behavior of the influenza virus resembled that of phages PR772 and Φ6, while the behavior of NDV was closer to that of phages MS2 and ΦX174. These results provide critical information for the selection of appropriate phage models to mimic the behavior of specific human and animal viruses in aerosols. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Comparison of Five Bacteriophages as Models for Viral Aerosol Studies

PubMed Central

Turgeon, Nathalie; Toulouse, Marie-Josée; Martel, Bruno; Moineau, Sylvain

2014-01-01

Bacteriophages are perceived to be good models for the study of airborne viruses because they are safe to use, some of them display structural features similar to those of human and animal viruses, and they are relatively easy to produce in large quantities. Yet, only a few studies have investigated them as models. It has previously been demonstrated that aerosolization, environmental conditions, and sampling conditions affect viral infectivity, but viral infectivity is virus dependent. Thus, several virus models are likely needed to study their general behavior in aerosols. The aim of this study was to compare the effects of aerosolization and sampling on the infectivity of five tail-less bacteriophages and two pathogenic viruses: MS2 (a single-stranded RNA [ssRNA] phage of the Leviviridae family), Φ6 (a segmented double-stranded RNA [dsRNA] phage of the Cystoviridae family), ΦX174 (a single-stranded DNA [ssDNA] phage of the Microviridae family), PM2 (a double-stranded DNA [dsDNA] phage of the Corticoviridae family), PR772 (a dsDNA phage of the Tectiviridae family), human influenza A virus H1N1 (an ssRNA virus of the Orthomyxoviridae family), and the poultry virus Newcastle disease virus (NDV; an ssRNA virus of the Paramyxoviridae family). Three nebulizers and two nebulization salt buffers (with or without organic fluid) were tested, as were two aerosol sampling devices, a liquid cyclone (SKC BioSampler) and a dry cyclone (National Institute for Occupational Safety and Health two-stage cyclone bioaerosol sampler). The presence of viruses in collected air samples was detected by culture and quantitative PCR (qPCR). Our results showed that these selected five phages behave differently when aerosolized and sampled. RNA phage MS2 and ssDNA phage ΦX174 were the most resistant to aerosolization and sampling. The presence of organic fluid in the nebulization buffer protected phages PR772 and Φ6 throughout the aerosolization and sampling with dry cyclones. In this experimental setup, the behavior of the influenza virus resembled that of phages PR772 and Φ6, while the behavior of NDV was closer to that of phages MS2 and ΦX174. These results provide critical information for the selection of appropriate phage models to mimic the behavior of specific human and animal viruses in aerosols. PMID:24795379

Diagnostic accuracy and applicability of a PCR system for the detection of Schistosoma mansoni DNA in human urine samples from an endemic area.

PubMed

Enk, Martin Johannes; Oliveira e Silva, Guilherme; Rodrigues, Nilton Barnabé

2012-01-01

Schistosomiasis caused by Schistosoma mansoni, one of the most neglected human parasitoses in Latin America and Africa, is routinely confirmed by microscopic visualization of eggs in stool. The main limitation of this diagnostic approach is its lack of sensitivity in detecting individual low worm burdens and consequently data on infection rates in low transmission settings are little reliable. According to the scientific literature, PCR assays are characterized by high sensitivity and specificity in detecting parasite DNA in biological samples. A simple and cost effective extraction method for DNA of Schistosoma mansoni from urine samples in combination with a conventional PCR assay was developed and applied in an endemic area. This urine based PCR system was tested for diagnostic accuracy among a population of a small village in an endemic area, comparing it to a reference test composed of three different parasitological techniques. The diagnostic parameters revealed a sensitivity of 100%, a specificity of 91.20%, positive and negative predictive values of 86.25% and 100%, respectively, and a test accuracy of 94.33%. Further statistical analysis showed a k index of 0.8806, indicating an excellent agreement between the reference test and the PCR system. Data obtained from the mouse model indicate the infection can be detected one week after cercariae penetration, opening a new perspective for early detection and patient management during this stage of the disease. The data indicate that this innovative PCR system provides a simple to handle and robust diagnostic tool for the detection of S. mansoni DNA from urine samples and a promising approach to overcome the diagnostic obstacles in low transmission settings. Furthermore the principals of this molecular technique, based on the examination of human urine samples may be useful for the diagnosis of other neglected tropical diseases that can be detected by trans-renal DNA.

Microbial Degradation of Forensic Samples of Biological Origin: Potential Threat to Human DNA Typing.

PubMed

Dash, Hirak Ranjan; Das, Surajit

2018-02-01

Forensic biology is a sub-discipline of biological science with an amalgam of other branches of science used in the criminal justice system. Any nucleated cell/tissue harbouring DNA, either live or dead, can be used as forensic exhibits, a source of investigation through DNA typing. These biological materials of human origin are rich source of proteins, carbohydrates, lipids, trace elements as well as water and, thus, provide a virtuous milieu for the growth of microbes. The obstinate microbial growth augments the degradation process and is amplified with the passage of time and improper storage of the biological materials. Degradation of these biological materials carriages a huge challenge in the downstream processes of forensic DNA typing technique, such as short tandem repeats (STR) DNA typing. Microbial degradation yields improper or no PCR amplification, heterozygous peak imbalance, DNA contamination from non-human sources, degradation of DNA by microbial by-products, etc. Consequently, the most precise STR DNA typing technique is nullified and definite opinion can be hardly given with degraded forensic exhibits. Thus, suitable precautionary measures should be taken for proper storage and processing of the biological exhibits to minimize their decaying process by micro-organisms.

Molecular Identification of Hookworm Isolates in Humans, Dogs and Soil in a Tribal Area in Tamil Nadu, India.

PubMed

George, Santosh; Levecke, Bruno; Kattula, Deepthi; Velusamy, Vasanthakumar; Roy, Sheela; Geldhof, Peter; Sarkar, Rajiv; Kang, Gagandeep

2016-08-01

Hookworms (Necator americanus and Ancylostoma duodenale) remain a major public health problem worldwide. Infections with hookworms (e.g., A. caninum, A. ceylanicum and A. braziliense) are also prevalent in dogs, but the role of dogs as a reservoir for zoonotic hookworm infections in humans needs to be further explored. As part of an open-label community based cluster-randomized trial in a tribal area in Tamil Nadu (India; 2013-2015), a total of 143 isolates of hookworm eggs from human stool were speciated based on a previously described PCR-RFLP methodology. The presence of hookworm DNA was confirmed in 119 of 143 human samples. N. americanus (100%) was the most prevalent species, followed by A. caninum (16.8%) and A. duodenale (8.4%). Because of the high prevalence of A. caninum in humans, dog samples were also collected to assess the prevalence of A. caninum in dogs. In 68 out of 77 canine stool samples the presence of hookworms was confirmed using PCR-RFLP. In dogs, both A. caninum (76.4%) and A. ceylanicum (27.9%) were identified. Additionally, to determine the contamination of soil with zoonotic hookworm larvae, topsoil was collected from defecating areas. Hookworm DNA was detected in 72 out of 78 soil samples that revealed presence of hookworm-like nematode larvae. In soil, different hookworm species were identified, with animal hookworms being more prevalent (A. ceylanicum: 60.2%, A. caninum: 29.4%, A. duodenale: 16.6%, N. americanus: 1.4%, A. braziliense: 1.4%). In our study we regularly detected the presence of A. caninum DNA in the stool of humans. Whether this is the result of infection is currently unknown but it does warrant a closer look at dogs as a potential reservoir.

Incidence of Echinococcus granulosus in Domestic Dogs in Palestine as Revealed by Copro-PCR

PubMed Central

Al-Jawabreh, Amer; Dumaidi, Kamal; Ereqat, Suheir; Nasereddin, Abedelmajeed; Al-Jawabreh, Hanan; Azmi, Kifaya; Al-Laham, Nahed; Abdeen, Ziad

2015-01-01

Hydatidosis or echinococcosisis considered a neglected zoonotic disease despite its high burden in the livestock industry and the high risk of infection by humans in endemic areas. In a cross-sectional study we estimated the copro-Incidence and also genotyped Echinococcus granulosus isolates from domestic dogs using polymerase chain reaction (PCR). Medical archives in nine major hospitals in Palestine were reviewed to determine incidence of E. granulosus infection detected in humans during surgery. Faecal samples were collected from 93 domestic dogs in three districts with the highest number of human cases: Al-Khalil (Hebron), Tubas and Jenin. Genomic DNA was extracted from dog faecal samples and amplified by PCR targeting the repeat DNA sequence (EgG1 Hae III) followed by sequencing of five positive samples. Genotyping was determined by sequencing and BLAST searching of mitochondrial cytochrome c oxidase subunit (CO1). The incidence of E. granulosus infection detected in humans at surgery was 1.2 per 100,000 in the West Bank and 1.0 per 100,000 in Gaza Strip. Seventeen of 93 domestic dogs (18%) were positive, based upon comparison with the Echinococcus DNA control. The five sequenced samples were confirmed to be E. granulosus. Successfully genotyped sample belonged to E.granulosus sensu stricto (formerly G1-G3 complex, sheep strain). For domestic dogs, age group (13-24 months) and sex were identified as two risk factors for contracting E. granulosus. The study identified the high incidence of E. granulosus sensu stricto in dogs in Palestine. PMID:26181591

Microfluidic DNA sample preparation method and device

DOEpatents

Krulevitch, Peter A.; Miles, Robin R.; Wang, Xiao-Bo; Mariella, Raymond P.; Gascoyne, Peter R. C.; Balch, Joseph W.

2002-01-01

Manipulation of DNA molecules in solution has become an essential aspect of genetic analyses used for biomedical assays, the identification of hazardous bacterial agents, and in decoding the human genome. Currently, most of the steps involved in preparing a DNA sample for analysis are performed manually and are time, labor, and equipment intensive. These steps include extraction of the DNA from spores or cells, separation of the DNA from other particles and molecules in the solution (e.g. dust, smoke, cell/spore debris, and proteins), and separation of the DNA itself into strands of specific lengths. Dielectrophoresis (DEP), a phenomenon whereby polarizable particles move in response to a gradient in electric field, can be used to manipulate and separate DNA in an automated fashion, considerably reducing the time and expense involved in DNA analyses, as well as allowing for the miniaturization of DNA analysis instruments. These applications include direct transport of DNA, trapping of DNA to allow for its separation from other particles or molecules in the solution, and the separation of DNA into strands of varying lengths.

Detection of human papillomavirus DNA in patients referred to a family practice colposcopy clinic.

PubMed

Holman, J R

1996-01-01

Human papillomavirus (HPV) is strongly implicated in the pathogenesis of cervical neoplasia. The ability of a commercially available kit (Virapap/Viratype) to detect evidence of HPV is compared with cervical cytology, colposcopy, and directed biopsies. During a period of 16 months, cervical samples from 241 consecutive new patients referred for a colposcopy examination were obtained for HPV-DNA hybridization typing according to the kit instructions. Samples were sent to a reference laboratory for testing. The results were compared with results of the colposcopy examination, cervical cytology, and directed cervical biopsy samples processed and evaluated by our hospital laboratory. HPV DNA was detected in 27 of 107 patients who had abnormal colposcopy findings for a sensitivity of 25 +/- 7.5 percent at the 90 percent confidence interval. One of 134 patients with normal findings was positive for a specificity of 99 +/- 5 percent at the 95 percent confidence interval. Based on a 75 percent probability of HPV in the population, the positive predictive value was 99 percent and the negative predictive value 30 percent. With the low negative predictive value and sensitivity, HPV-DNA testing by this commercial kit is not an adequate tool for screening HPV in this population.

Label-Free Nanopore Biosensor for Rapid and Highly Sensitive Cocaine Detection in Complex Biological Fluids.

PubMed

Rauf, Sana; Zhang, Ling; Ali, Asghar; Liu, Yang; Li, Jinghong

2017-02-24

Detection of very low amounts of illicit drugs such as cocaine in clinical fluids like serum continues to be important for many areas in the fight against drug trafficking. Herein, we constructed a label-free nanopore biosensor for rapid and highly sensitive detection of cocaine in human serum and saliva samples based on target-induced strand release strategy. In this bioassay, an aptamer for cocaine was prehybridized with a short complementary DNA. Owing to cocaine specific binding with aptamer, the short DNA strand was displaced from aptamer and translocation of this output DNA through α-hemolysin nanopore generated distinct spike-like current blockages. When plotted in double-logarithmic scale, a linear relationship between target cocaine concentration and output DNA event frequency was obtained in a wide concentration range from 50 nM to 100 μM of cocaine, with the limit of detection down to 50 nM. In addition, this aptamer-based sensor method was successfully applied for cocaine detection in complex biological fluids like human saliva and serum samples with great selectivity. Simple preparation, low cost, rapid, label-free, and real sample detection are the motivating factors for practical application of the proposed biosensor.

TriXY-Homogeneous genetic sexing of highly degraded forensic samples including hair shafts.

PubMed

Madel, Maria-Bernadette; Niederstätter, Harald; Parson, Walther

2016-11-01

Sexing of biological evidence is an important aspect in forensic investigations. A routinely used molecular-genetic approach to this endeavour is the amelogenin sex test, which is integrated in most commercially available polymerase chain reaction (PCR) kits for human identification. However, this assay is not entirely effective in respect to highly degraded DNA samples. This study presents a homogeneous PCR assay for robust sex diagnosis, especially for the analysis of severely fragmented DNA. The introduced triplex for the X and Y chromosome (TriXY) is based on real-time PCR amplification of short intergenic sequences (<50bp) on both gonosomes. Subsequent PCR product examination and molecular-genetic sex-assignment rely on high-resolution melting (HRM) curve analysis. TriXY was optimized using commercially available multi-donor human DNA preparations of either male or female origin and successfully evaluated on challenging samples, including 46 ancient DNA specimens from archaeological excavations and a total of 16 DNA samples extracted from different segments of eight hair shafts of male and female donors. Additionally, sensitivity and cross-species amplification were examined to further test the assay's utility in forensic investigations. TriXY's closed-tube format avoids post-PCR sample manipulations and, therefore, distinctly reduces the risk of PCR product carry-over contamination and sample mix-up, while reducing labour and financial expenses at the same time. The method is sensitive down to the DNA content of approximately two diploid cells and has proven highly useful on severely fragmented and low quantity ancient DNA samples. Furthermore, it even allowed for sexing of proximal hair shafts with very good results. In summary, TriXY facilitates highly sensitive, rapid, and costeffective genetic sex-determination. It outperforms existing sexing methods both in terms of sensitivity and minimum required template molecule lengths. Therefore, we feel confident that TriXY will prove to be a reliable addition to the toolbox currently used for sex-typing in forensic genetics and other fields of research. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

DNA preservation in silk.

PubMed

Liu, Yawen; Zheng, Zhaozhu; Gong, He; Liu, Meng; Guo, Shaozhe; Li, Gang; Wang, Xiaoqin; Kaplan, David L

2017-06-27

The structure of DNA is susceptible to alterations at high temperature and on changing pH, irradiation and exposure to DNase. Options to protect and preserve DNA during storage are important for applications in genetic diagnosis, identity authentication, drug development and bioresearch. In the present study, the stability of total DNA purified from human dermal fibroblast cells, as well as that of plasmid DNA, was studied in silk protein materials. The DNA/silk mixtures were stabilized on filter paper (silk/DNA + filter) or filter paper pre-coated with silk and treated with methanol (silk/DNA + PT-filter) as a route to practical utility. After air-drying and water extraction, 50-70% of the DNA and silk could be retrieved and showed a single band on electrophoretic gels. 6% silk/DNA + PT-filter samples provided improved stability in comparison with 3% silk/DNA + filter samples and DNA + filter samples for DNA preservation, with ∼40% of the band intensity remaining at 37 °C after 40 days and ∼10% after exposure to UV light for 10 hours. Quantitative analysis using the PicoGreen assay confirmed the results. The use of Tris/borate/EDTA (TBE) buffer enhanced the preservation and/or extraction of the DNA. The DNA extracted after storage maintained integrity and function based on serving as a functional template for PCR amplification of the gene for zinc finger protein 750 (ZNF750) and for transgene expression of red fluorescence protein (dsRed) in HEK293 cells. The high molecular weight and high content of a crystalline beta-sheet structure formed on the coated surfaces likely accounted for the preservation effects observed for the silk/DNA + PT-filter samples. Although similar preservation effects were also obtained for lyophilized silk/DNA samples, the rapid and simple processing available with the silk-DNA-filter membrane system makes it appealing for future applications.

Detection of human papillomavirus in normal oral cavity in a group of Pakistani subjects using real-time PCR.

PubMed

Gichki, Abdul Samad; Buajeeb, Waranun; Doungudomdacha, Sombhun; Khovidhunkit, Siribang-on Pibooniyom

2012-01-01

Since there is evidence that human papillomavirus (HPV) may play some role in oral carcinogenesis, we investigated the presence of HPV in a group of Pakistani subjects with normal oral cavity using real-time PCR analysis. Two-hundred patients attending the Dental Department, Sandaman Provincial Hospital, Balochistan, Pakistan, were recruited. After interview, oral epithelial cells were collected by scraping and subjected to DNA extraction. The HPV-positive DNA samples were further analyzed using primer sets specific for HPV-16 and -18. It was found that out of 200 DNA samples, 192 were PCR-positive for the β-globin gene and these were subsequently examined for the presence of HPV DNA. Among these, 47 (24.5%) were HPV-positive with the virus copy number ranged between 0.43-32 copies per 1 μg of total DNA (9-99 copies per PCR reaction). There were 4 and 11 samples containing HPV-16 and -18, respectively. Additionally, one sample harbored both types of HPV. Among the investigated clinical parameters, smoking habit was associated with the presence of HPV (p=0.001) while others indicated no significant association. The prevalence of HPV in normal oral cavity in our Pakistani subjects appears to be comparable to other studies. However, the association between the presence of HPV and smoking warrants further investigations whether both of these factors can cooperate in inducing oral cancer in this group of patients.

DNA and bone structure preservation in medieval human skeletons.

PubMed

Coulson-Thomas, Yvette M; Norton, Andrew L; Coulson-Thomas, Vivien J; Florencio-Silva, Rinaldo; Ali, Nadir; Elmrghni, Samir; Gil, Cristiane D; Sasso, Gisela R S; Dixon, Ronald A; Nader, Helena B

2015-06-01

Morphological and ultrastructural data from archaeological human bones are scarce, particularly data that have been correlated with information on the preservation of molecules such as DNA. Here we examine the bone structure of macroscopically well-preserved medieval human skeletons by transmission electron microscopy and immunohistochemistry, and the quantity and quality of DNA extracted from these skeletons. DNA technology has been increasingly used for analyzing physical evidence in archaeological forensics; however, the isolation of ancient DNA is difficult since it is highly degraded, extraction yields are low and the co-extraction of PCR inhibitors is a problem. We adapted and optimised a method that is frequently used for isolating DNA from modern samples, Chelex(®) 100 (Bio-Rad) extraction, for isolating DNA from archaeological human bones and teeth. The isolated DNA was analysed by real-time PCR using primers targeting the sex determining region on the Y chromosome (SRY) and STR typing using the AmpFlSTR(®) Identifiler PCR Amplification kit. Our results clearly show the preservation of bone matrix in medieval bones and the presence of intact osteocytes with well preserved encapsulated nuclei. In addition, we show how effective Chelex(®) 100 is for isolating ancient DNA from archaeological bones and teeth. This optimised method is suitable for STR typing using kits aimed specifically at degraded and difficult DNA templates since amplicons of up to 250bp were successfully amplified. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Assessment of the role of DNA repair in damaged forensic samples.

PubMed

Ambers, Angie; Turnbough, Meredith; Benjamin, Robert; King, Jonathan; Budowle, Bruce

2014-11-01

Previous studies on DNA damage and repair have involved in vitro laboratory procedures that induce a single type of lesion in naked templates. Although repair of singular, sequestered types of DNA damage has shown some success, forensic and ancient specimens likely contain a number of different types of lesions. This study sought to (1) develop protocols to damage DNA in its native state, (2) generate a pool of candidate samples for repair that more likely emulate authentic forensic samples, and (3) assess the ability of the PreCR(TM) Repair Mix to repair the resultant lesions. Complexed, native DNA is more difficult to damage than naked DNA. Modified procedures included the use of higher concentrations and longer exposure times. Three types of samples, those that demonstrated damage based on short tandem repeat (STR) profile signals, were selected for repair experiments: environmentally damaged bloodstains, bleach-damaged whole blood, and human skeletal remains. Results showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR(TM) assay. The data suggest that the use of PreCR in casework should be considered with caution due to the assay's varied results.

Improved Y-STR typing for disaster victim identification, missing persons investigations, and historical human skeletal remains.

PubMed

Ambers, Angie; Votrubova, Jitka; Vanek, Daniel; Sajantila, Antti; Budowle, Bruce

2018-02-23

Bones are a valuable source of DNA in forensic, anthropological, and archaeological investigations. There are a number of scenarios in which the only samples available for testing are highly degraded and/or skeletonized. Often it is necessary to perform more than one type of marker analysis on such samples in order to compile sufficient data for identification. Lineage markers, such as Y-STRs and mitochondrial DNA (mtDNA), represent important systems to complement autosomal DNA markers and anthropological metadata in making associations between unidentified remains and living relatives or for characterization of the remains for historical and archaeological studies. In this comparative study, Y-STR typing with both Yfiler™ and Yfiler™ Plus (Thermo Fisher Scientific, Waltham, MA, USA) was performed on a variety of human skeletal remains, including samples from the American Civil War (1861-1865), the late nineteenth century gold rush era in Deadwood, SD, USA (1874-1877), the Seven Years' War (1756-1763), a seventeenth-century archaeological site in Raspenava, Bohemia (Czech Republic), and World War II (1939-1945). The skeletal remains used for this study were recovered from a wide range of environmental conditions and were extracted using several common methods. Regardless of the DNA extraction method used and the age/condition of the remains, 22 out of 24 bone samples yielded a greater number of alleles using the Yfiler™ Plus kit compared to the Yfiler™ kit using the same quantity of input DNA. There was no discernable correlation with the degradation index values for these samples. Overall, the efficacy of the Yfiler™ Plus assay was demonstrated on degraded DNA from skeletal remains. Yfiler™ Plus increases the discriminatory power over the previous generation multiplex due to the larger set of Y-STR markers available for analysis and buffer modifications with the newer version kit. Increased haplotype resolution is provided to infer or refute putative genetic relationships.

Bona fide colour: DNA prediction of human eye and hair colour from ancient and contemporary skeletal remains

PubMed Central

2013-01-01

Background DNA analysis of ancient skeletal remains is invaluable in evolutionary biology for exploring the history of species, including humans. Contemporary human bones and teeth, however, are relevant in forensic DNA analyses that deal with the identification of perpetrators, missing persons, disaster victims or family relationships. They may also provide useful information towards unravelling controversies that surround famous historical individuals. Retrieving information about a deceased person’s externally visible characteristics can be informative in both types of DNA analyses. Recently, we demonstrated that human eye and hair colour can be reliably predicted from DNA using the HIrisPlex system. Here we test the feasibility of the novel HIrisPlex system at establishing eye and hair colour of deceased individuals from skeletal remains of various post-mortem time ranges and storage conditions. Methods Twenty-one teeth between 1 and approximately 800 years of age and 5 contemporary bones were subjected to DNA extraction using standard organic protocol followed by analysis using the HIrisPlex system. Results Twenty-three out of 26 bone DNA extracts yielded the full 24 SNP HIrisPlex profile, therefore successfully allowing model-based eye and hair colour prediction. HIrisPlex analysis of a tooth from the Polish general Władysław Sikorski (1881 to 1943) revealed blue eye colour and blond hair colour, which was positively verified from reliable documentation. The partial profiles collected in the remaining three cases (two contemporary samples and a 14th century sample) were sufficient for eye colour prediction. Conclusions Overall, we demonstrate that the HIrisPlex system is suitable, sufficiently sensitive and robust to successfully predict eye and hair colour from ancient and contemporary skeletal remains. Our findings, therefore, highlight the HIrisPlex system as a promising tool in future routine forensic casework involving skeletal remains, including ancient DNA studies, for the prediction of eye and hair colour of deceased individuals. PMID:23317428

Bona fide colour: DNA prediction of human eye and hair colour from ancient and contemporary skeletal remains.

PubMed

Draus-Barini, Jolanta; Walsh, Susan; Pośpiech, Ewelina; Kupiec, Tomasz; Głąb, Henryk; Branicki, Wojciech; Kayser, Manfred

2013-01-14

DNA analysis of ancient skeletal remains is invaluable in evolutionary biology for exploring the history of species, including humans. Contemporary human bones and teeth, however, are relevant in forensic DNA analyses that deal with the identification of perpetrators, missing persons, disaster victims or family relationships. They may also provide useful information towards unravelling controversies that surround famous historical individuals. Retrieving information about a deceased person's externally visible characteristics can be informative in both types of DNA analyses. Recently, we demonstrated that human eye and hair colour can be reliably predicted from DNA using the HIrisPlex system. Here we test the feasibility of the novel HIrisPlex system at establishing eye and hair colour of deceased individuals from skeletal remains of various post-mortem time ranges and storage conditions. Twenty-one teeth between 1 and approximately 800 years of age and 5 contemporary bones were subjected to DNA extraction using standard organic protocol followed by analysis using the HIrisPlex system. Twenty-three out of 26 bone DNA extracts yielded the full 24 SNP HIrisPlex profile, therefore successfully allowing model-based eye and hair colour prediction. HIrisPlex analysis of a tooth from the Polish general Władysław Sikorski (1881 to 1943) revealed blue eye colour and blond hair colour, which was positively verified from reliable documentation. The partial profiles collected in the remaining three cases (two contemporary samples and a 14th century sample) were sufficient for eye colour prediction. Overall, we demonstrate that the HIrisPlex system is suitable, sufficiently sensitive and robust to successfully predict eye and hair colour from ancient and contemporary skeletal remains. Our findings, therefore, highlight the HIrisPlex system as a promising tool in future routine forensic casework involving skeletal remains, including ancient DNA studies, for the prediction of eye and hair colour of deceased individuals.

Ubiquity and Diversity of Human-Associated Demodex Mites

PubMed Central

Thoemmes, Megan S.; Fergus, Daniel J.; Urban, Julie; Trautwein, Michelle; Dunn, Robert R.

2014-01-01

Demodex mites are a group of hair follicle and sebaceous gland-dwelling species. The species of these mites found on humans are arguably the animals with which we have the most intimate interactions. Yet, their prevalence and diversity have been poorly explored. Here we use a new molecular method to assess the occurrence of Demodex mites on humans. In addition, we use the 18S rRNA gene (18S rDNA) to assess the genetic diversity and evolutionary history of Demodex lineages. Within our samples, 100% of people over 18 years of age appear to host at least one Demodex species, suggesting that Demodex mites may be universal associates of adult humans. A phylogenetic analysis of 18S rDNA reveals intraspecific structure within one of the two named human-associated Demodex species, D. brevis. The D. brevis clade is geographically structured, suggesting that new lineages are likely to be discovered as humans from additional geographic regions are sampled. PMID:25162399

Evaluation of Interindividual Human Variation in Bioactivation and DNA Adduct Formation of Estragole in Liver Predicted by Physiologically Based Kinetic/Dynamic and Monte Carlo Modeling.

PubMed

Punt, Ans; Paini, Alicia; Spenkelink, Albertus; Scholz, Gabriele; Schilter, Benoit; van Bladeren, Peter J; Rietjens, Ivonne M C M

2016-04-18

Estragole is a known hepatocarcinogen in rodents at high doses following metabolic conversion to the DNA-reactive metabolite 1'-sulfooxyestragole. The aim of the present study was to model possible levels of DNA adduct formation in (individual) humans upon exposure to estragole. This was done by extending a previously defined PBK model for estragole in humans to include (i) new data on interindividual variation in the kinetics for the major PBK model parameters influencing the formation of 1'-sulfooxyestragole, (ii) an equation describing the relationship between 1'-sulfooxyestragole and DNA adduct formation, (iii) Monte Carlo modeling to simulate interindividual human variation in DNA adduct formation in the population, and (iv) a comparison of the predictions made to human data on DNA adduct formation for the related alkenylbenzene methyleugenol. Adequate model predictions could be made, with the predicted DNA adduct levels at the estimated daily intake of estragole of 0.01 mg/kg bw ranging between 1.6 and 8.8 adducts in 10(8) nucleotides (nts) (50th and 99th percentiles, respectively). This is somewhat lower than values reported in the literature for the related alkenylbenzene methyleugenol in surgical human liver samples. The predicted levels seem to be below DNA adduct levels that are linked with tumor formation by alkenylbenzenes in rodents, which were estimated to amount to 188-500 adducts per 10(8) nts at the BMD10 values of estragole and methyleugenol. Although this does not seem to point to a significant health concern for human dietary exposure, drawing firm conclusions may have to await further validation of the model's predictions.

Assessing the performance of a Loop Mediated Isothermal Amplification (LAMP) assay for the detection and subtyping of high-risk suptypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) without DNA purification.

PubMed

Rohatensky, Mitchell G; Livingstone, Devon M; Mintchev, Paul; Barnes, Heather K; Nakoneshny, Steven C; Demetrick, Douglas J; Dort, Joseph C; van Marle, Guido

2018-02-08

Oropharyngeal Squamous Cell Carcinoma (OPSCC) is increasing in incidence despite a decline in traditional risk factors. Human Papilloma Virus (HPV), specifically subtypes 16, 18, 31 and 35, has been implicated as the high-risk etiologic agent. HPV positive cancers have a significantly better prognosis than HPV negative cancers of comparable stage, and may benefit from different treatment regimens. Currently, HPV related carcinogenesis is established indirectly through Immunohistochemistry (IHC) staining for p16, a tumour suppressor gene, or polymerase chain reaction (PCR) that directly tests for HPV DNA in biopsied tissue. Loop mediated isothermal amplification (LAMP) is more accurate than IHC, more rapid than PCR and is significantly less costly. In previous work we showed that a subtype specific HPV LAMP assay performed similar to PCR on purified DNA. In this study we examined the performance of this LAMP assay without DNA purification. We used LAMP assays using established primers for HPV 16 and 18, and new primers for HPV 31 and 35. LAMP reaction conditions were tested on serial dilutions of plasmid HPV DNA to confirm minimum viral copy number detection thresholds. LAMP was then performed directly on different human cell line samples without DNA purification. Our LAMP assays could detect 10 5 , 10 3 , 10 4 , and 10 5 copies of plasmid DNA for HPV 16, 18, 31, and 35, respectively. All primer sets were subtype specific, with no cross-amplification. Our LAMP assays also reliably amplified subtype specific HPV DNA from samples without requiring DNA isolation and purification. The high risk OPSCC HPV subtype specific LAMP primer sets demonstrated, excellent clinically relevant, minimum copy number detection thresholds with an easy readout system. Amplification directly from samples without purification illustrated the robust nature of the assay, and the primers used. This lends further support HPV type specific LAMP assays, and these specific primer sets and assays can be further developed to test for HPV in OPSCC in resource and lab limited settings, or even bedside testing.

Automation of DNA and miRNA co-extraction for miRNA-based identification of human body fluids and tissues.

PubMed

Kulstein, Galina; Marienfeld, Ralf; Miltner, Erich; Wiegand, Peter

2016-10-01

In the last years, microRNA (miRNA) analysis came into focus in the field of forensic genetics. Yet, no standardized and recommendable protocols for co-isolation of miRNA and DNA from forensic relevant samples have been developed so far. Hence, this study evaluated the performance of an automated Maxwell® 16 System-based strategy (Promega) for co-extraction of DNA and miRNA from forensically relevant (blood and saliva) samples compared to (semi-)manual extraction methods. Three procedures were compared on the basis of recovered quantity of DNA and miRNA (as determined by real-time PCR and Bioanalyzer), miRNA profiling (shown by Cq values and extraction efficiency), STR profiles, duration, contamination risk and handling. All in all, the results highlight that the automated co-extraction procedure yielded the highest miRNA and DNA amounts from saliva and blood samples compared to both (semi-)manual protocols. Also, for aged and genuine samples of forensically relevant traces the miRNA and DNA yields were sufficient for subsequent downstream analysis. Furthermore, the strategy allows miRNA extraction only in cases where it is relevant to obtain additional information about the sample type. Besides, this system enables flexible sample throughput and labor-saving sample processing with reduced risk of cross-contamination. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Occurrence and removal efficiency of parasitic protozoa in Swedish wastewater treatment plants.

PubMed

Berglund, Björn; Dienus, Olaf; Sokolova, Ekaterina; Berglind, Emma; Matussek, Andreas; Pettersson, Thomas; Lindgren, Per-Eric

2017-11-15

Giardia intestinalis, Cryptosporidium spp., Entamoeba histolytica and Dientamoeba fragilis are parasitic protozoa and causative agents of gastroenteritis in humans. G. intestinalis and Cryptosporidium spp. in particular are the most common protozoa associated with waterborne outbreaks in high-income countries. Surveillance of protozoan prevalence in wastewater and evaluation of wastewater treatment removal efficiencies of protozoan pathogens is therefore imperative for assessment of human health risk. In this study, influent and effluent wastewater samples from three wastewater treatment plants in Sweden were collected over nearly one year and assessed for prevalence of parasitic protozoa. Quantitative real-time PCR using primers specific for the selected protozoa Cryptosporidium spp., G. intestinalis, E. histolytica, Entamoeba dispar and D. fragilis was used for protozoan DNA detection and assessment of wastewater treatment removal efficiencies. Occurrence of G. intestinalis, E. dispar and D. fragilis DNA was assessed in both influent (44, 30 and 39 out of 51 samples respectively) and effluent wastewater (14, 9 and 33 out of 51 samples respectively) in all three wastewater treatment plants. Mean removal efficiencies of G. intestinalis, E. dispar and D. fragilis DNA quantities, based on all three wastewater treatment plants studied varied between 67 and 87%, 37-75% and 20-34% respectively. Neither E. histolytica nor Cryptosporidium spp. were detected in any samples. Overall, higher quantities of protozoan DNA were observed from February to June 2012. The high prevalence of protozoa in influent wastewater indicates the need for continued monitoring of these pathogens in wastewater-associated aquatic environments to minimise the potential risk for human infection. Copyright © 2017 Elsevier B.V. All rights reserved.

Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts

PubMed Central

Thierry, Alain R.; Mouliere, Florent; Gongora, Celine; Ollier, Jeremy; Robert, Bruno; Ychou, Marc; Del Rio, Maguy; Molina, Franck

2010-01-01

Although circulating DNA (ctDNA) could be an attractive tool for early cancer detection, diagnosis, prognosis, monitoring or prediction of response to therapies, knowledge on its origin, form and rate of release is poor and often contradictory. Here, we describe an experimental system to systematically examine these aspects. Nude mice were xenografted with human HT29 or SW620 colorectal carcinoma (CRC) cells and ctDNA was analyzed by Q–PCR with highly specific and sensitive primer sets at different times post-graft. We could discriminate ctDNA from normal (murine) cells and from mutated and non-mutated tumor (human) cells by using species-specific KRAS or PSAT1 primers and by assessing the presence of the BRAF V600E mutation. The concentration of human (mutated and non-mutated) ctDNA increased significantly with tumor growth. Conversely, and differently from previous studies, low, constant level of mouse ctDNA was observed, thus facilitating the study of mutated and non-mutated tumor derived ctDNA. Finally, analysis of ctDNA fragmentation confirmed the predominance of low-size fragments among tumor ctDNA from mice with bigger tumors. Higher ctDNA fragmentation was also observed in plasma samples from three metastatic CRC patients in comparison to healthy individuals. Our data confirm the predominance of mononucleosome-derived fragments in plasma from xenografted animals and, as a consequence, of apoptosis as a source of ctDNA, in particular for tumor-derived ctDNA. Altogether, our results suggest that ctDNA features vary during CRC tumor development and our experimental system might be a useful tool to follow such variations. PMID:20494973

Absence of bacterial DNA in culture-negative urine from cats with and without lower urinary tract disease.

PubMed

Lund, Heidi Sjetne; Skogtun, Gaute; Sørum, Henning; Eggertsdóttir, Anna Vigdís

2015-10-01

A diagnosis of bacterial cystitis commonly relies on a positive microbiological culture demonstrating the presence of a significant number of colony-forming units/ml urine, as urine within the upper urinary tract, bladder and proximal urethra generally is considered sterile. Recent studies from human and veterinary medicine indicate the presence of non-culturable bacteria in culture-negative urine samples. The aim of the present study was to determine the occurrence of bacterial DNA in culture-negative urine samples from cats with signs of feline lower urinary tract disease (FLUTD) and healthy control cats by 16S ribosomal DNA PCR and subsequent sequencing. The study sample included 38 culture-negative urine samples from cats with FLUTD and 43 culture-negative samples from control cats. Eight culture-positive urine samples from cats with FLUTD were included as external positive controls in addition to negative reaction controls. Of possible methodological limitations, degradation of DNA due to storage, the use of non-sedimented urine for DNA isolation and lack of internal positive reaction controls should be mentioned. The positive controls were recognised, but occurrence of bacterial DNA in culture-negative urine from cats with or without signs of lower urinary tract disease was not demonstrated. However, considering the possible methodological limitations, the presence of bacterial DNA in the urine of culture-negative FLUTD cats cannot be excluded based on the present results alone. Therefore, a prospective study reducing the possibility of degradation of DNA due to storage, in combination with modifications enhancing the chance of detecting even lower levels of bacterial DNA in culture-negative samples, seems warranted. © ISFM and AAFP 2014.

Adenovirus 36 DNA in human adipose tissue.

PubMed

Ponterio, E; Cangemi, R; Mariani, S; Casella, G; De Cesare, A; Trovato, F M; Garozzo, A; Gnessi, L

2015-12-01

Recent studies have suggested a possible correlation between obesity and adenovirus 36 (Adv36) infection in humans. As information on adenoviral DNA presence in human adipose tissue are limited, we evaluated the presence of Adv36 DNA in adipose tissue of 21 adult overweight or obese patients. Total DNA was extracted from adipose tissue biopsies. Virus detection was performed using PCR protocols with primers against specific Adv36 fiber protein and the viral oncogenic E4orf1 protein nucleotide sequences. Sequences were aligned with the NCBI database and phylogenetic analyses were carried out with MEGA6 software. Adv36 DNA was found in four samples (19%). This study indicates that some individuals carry Adv36 in the visceral adipose tissue. Further studies are needed to determine the specific effect of Adv36 infection on adipocytes, the prevalence of Adv36 infection and its relationship with obesity in the perspective of developing a vaccine that could potentially prevent or mitigate infection.

Multiple human papillomavirus infections and type-competition in women from a clinic attendee population in China.

PubMed

Nie, Jianhui; Liu, Jianhua; Xie, Hui; Sun, Zhengrong; Zhao, Juan; Chen, Qingqing; Liu, Yangyang; Huang, Weijin; Ruan, Qiang; Wang, Youchun

2016-11-01

To investigate the multi-infection patterns and type competition for human papillomavirus (HPV) in Chinese clinic attending women and evaluate the association between the infection status and cancer risk. Three hundred and thirty-two HPV-DNA-positive samples were genotyped for 21 HPVs and tested for 13 types of HPV neutralizing antibody using pseudoviron-based assay. Odds ratios (ORs) were calculated to evaluate the coinfection patterns for both DNA and neutralizing antibody (NAb), and the associations of HSIL+ with HPV DNA and NAb. Of the 332 HPV-DNA-positive subjects, 279 (84.0%) were detected as NAb positive. Multi-positive results were identified in 23.2% (77/332) HPV DNA tests and 60.2% (168/279) HPV NAb assays. The NAb titers for the multiple-positive samples (geometric mean 214) were significantly higher than the single-positive samples (geometric mean 114) (P < 0.01). HPV16-HPV52 was identified as a type-competition pair for both HPV DNA (OR = 0.09; 95%CI = 0.02-0.42) and NAb (OR = 0.27; 95%CI = 0.11-0.69) data. Compared to other types, HPV16 DNA was associated significantly with high risk of HSIL+ (OR = 2.57; 95%CI = 1.23-5.38). HPV16-HPV52 was identified as a potential type-competition pair, which might result in the type modulation with the implementation of the approved bi- or quadri-valent vaccines. J. Med. Virol. 88:1989-1998, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Multiplexed SNP typing of ancient DNA clarifies the origin of Andaman mtDNA haplogroups amongst South Asian tribal populations.

PubMed

Endicott, Phillip; Metspalu, Mait; Stringer, Chris; Macaulay, Vincent; Cooper, Alan; Sanchez, Juan J

2006-12-20

The issue of errors in genetic data sets is of growing concern, particularly in population genetics where whole genome mtDNA sequence data is coming under increased scrutiny. Multiplexed PCR reactions, combined with SNP typing, are currently under-exploited in this context, but have the potential to genotype whole populations rapidly and accurately, significantly reducing the amount of errors appearing in published data sets. To show the sensitivity of this technique for screening mtDNA genomic sequence data, 20 historic samples of the enigmatic Andaman Islanders and 12 modern samples from three Indian tribal populations (Chenchu, Lambadi and Lodha) were genotyped for 20 coding region sites after provisional haplogroup assignment with control region sequences. The genotype data from the historic samples significantly revise the topologies for the Andaman M31 and M32 mtDNA lineages by rectifying conflicts in published data sets. The new Indian data extend the distribution of the M31a lineage to South Asia, challenging previous interpretations of mtDNA phylogeography. This genetic connection between the ancestors of the Andamanese and South Asian tribal groups approximately 30 kya has important implications for the debate concerning migration routes and settlement patterns of humans leaving Africa during the late Pleistocene, and indicates the need for more detailed genotyping strategies. The methodology serves as a low-cost, high-throughput model for the production and authentication of data from modern or ancient DNA, and demonstrates the value of museum collections as important records of human genetic diversity.

Multiplexed SNP Typing of Ancient DNA Clarifies the Origin of Andaman mtDNA Haplogroups amongst South Asian Tribal Populations

PubMed Central

Endicott, Phillip; Metspalu, Mait; Stringer, Chris; Macaulay, Vincent; Cooper, Alan; Sanchez, Juan J.

2006-01-01

The issue of errors in genetic data sets is of growing concern, particularly in population genetics where whole genome mtDNA sequence data is coming under increased scrutiny. Multiplexed PCR reactions, combined with SNP typing, are currently under-exploited in this context, but have the potential to genotype whole populations rapidly and accurately, significantly reducing the amount of errors appearing in published data sets. To show the sensitivity of this technique for screening mtDNA genomic sequence data, 20 historic samples of the enigmatic Andaman Islanders and 12 modern samples from three Indian tribal populations (Chenchu, Lambadi and Lodha) were genotyped for 20 coding region sites after provisional haplogroup assignment with control region sequences. The genotype data from the historic samples significantly revise the topologies for the Andaman M31 and M32 mtDNA lineages by rectifying conflicts in published data sets. The new Indian data extend the distribution of the M31a lineage to South Asia, challenging previous interpretations of mtDNA phylogeography. This genetic connection between the ancestors of the Andamanese and South Asian tribal groups ∼30 kya has important implications for the debate concerning migration routes and settlement patterns of humans leaving Africa during the late Pleistocene, and indicates the need for more detailed genotyping strategies. The methodology serves as a low-cost, high-throughput model for the production and authentication of data from modern or ancient DNA, and demonstrates the value of museum collections as important records of human genetic diversity. PMID:17218991

Effects of anticoagulant, processing delay, and assay method (branched DNA versus reverse transcriptase PCR) on measurement of human immunodeficiency virus type 1 RNA levels in plasma.

PubMed

Kirstein, L M; Mellors, J W; Rinaldo, C R; Margolick, J B; Giorgi, J V; Phair, J P; Dietz, E; Gupta, P; Sherlock, C H; Hogg, R; Montaner, J S; Muñoz, A

1999-08-01

We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (P < 0.05) but small after 6 h (bDNA assay, -0.12 log(10) copies/ml; RT-PCR, -0.05 log(10) copies/ml) and after 18 h (bDNA assay, -0.27 log(10) copies/ml; RT-PCR, -0.15 log(10) copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, -0.002 log(10) copies/ml; RT-PCR, -0.02 log(10) copies/ml), but it was after 18 h (bDNA assay, -0.09 log(10) copies/ml; RT-PCR, -0.09 log(10) copies/ml). Only 4% of samples processed after 6 h lost more than 50% (>/=0.3 log(10) copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4(+)-cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (-0.05 to -0.12 log(10) copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4(+)-cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP.

Xenobiotic metabolism induction and bulky DNA adducts generated by particulate matter pollution in BEAS-2B cell line: geographical and seasonal influence.

PubMed

Lepers, Capucine; André, Véronique; Dergham, Mona; Billet, Sylvain; Verdin, Anthony; Garçon, Guillaume; Dewaele, Dorothée; Cazier, Fabrice; Sichel, François; Shirali, Pirouz

2014-06-01

Airborne particulate matter (PM) toxicity is of growing interest as diesel exhaust particles have been classified as carcinogenic to humans. However, PM is a mixture of chemicals, and respective contribution of organic and inorganic fractions to PM toxicity remains unclear. Thus, we analysed the link between chemical composition of PM samples and bulky DNA adduct formation supported by CYP1A1 and 1B1 genes induction and catalytic activities. We used six native PM samples, collected in industrial, rural or urban areas, either during the summer or winter, and carried out our experiments on the human bronchial epithelial cell line BEAS-2B. Cell exposure to PM resulted in CYP1A1 and CYP1B1 genes induction. This was followed by an increase in EROD activity, leading to bulky DNA adduct formation in exposed cells. Bulky DNA adduct intensity was associated to global EROD activity, but this activity was poorly correlated with CYPs mRNA levels. However, EROD activity was correlated with both metal and polycyclic aromatic hydrocarbon (PAH) content. Finally, principal components analysis revealed three clusters for PM chemicals, and suggested synergistic effects of metals and PAHs on bulky DNA adduct levels. This study showed the ability of PM samples from various origins to generate bulky DNA adducts in BEAS-2B cells. This formation was promoted by increased expression and activity of CYPs involved in PAHs activation into reactive metabolites. However, our data highlight that bulky DNA adduct formation is only partly explained by PM content in PAHs, and suggest that inorganic compounds, such as iron, may promote bulky DNA adduct formation by supporting CYP activity. Copyright © 2013 John Wiley & Sons, Ltd.

Association of Global DNA Methylation and Global DNA Hydroxymethylation with Metals and Other Exposures in Human Blood DNA Samples

PubMed Central

Tang, Wan-yee; Shang, Yan; Umans, Jason G.; Francesconi, Kevin A.; Goessler, Walter; Ledesma, Marta; Leon, Montserrat; Laclaustra, Martin; Pollak, Jonathan; Guallar, Eliseo; Cole, Shelley A.; Fallin, M. Dani; Navas-Acien, Ana

2014-01-01

Background: The association between human blood DNA global methylation and global hydroxymethylation has not been evaluated in population-based studies. No studies have evaluated environmental determinants of global DNA hydroxymethylation, including exposure to metals. Objective: We evaluated the association between global DNA methylation and global DNA hydroxymethylation in 48 Strong Heart Study participants for which selected metals had been measured in urine at baseline and DNA was available from 1989–1991 (visit 1) and 1998–1999 (visit 3). Methods: We measured the percentage of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in samples using capture and detection antibodies followed by colorimetric quantification. We explored the association of participant characteristics (i.e., age, adiposity, smoking, and metal exposure) with both global DNA methylation and global DNA hydroxymethylation. Results: The Spearman’s correlation coefficient for 5-mC and 5-hmC levels was 0.32 (p = 0.03) at visit 1 and 0.54 (p < 0.001) at visit 3. Trends for both epigenetic modifications were consistent across potential determinants. In cross-sectional analyses, the odds ratios of methylated and hydroxymethylated DNA were 1.56 (95% CI: 0.95, 2.57) and 1.76 (95% CI: 1.07, 2.88), respectively, for the comparison of participants above and below the median percentage of dimethylarsinate. The corresponding odds ratios were 1.64 (95% CI: 1.02, 2.65) and 1.16 (95% CI: 0.70, 1.94), respectively, for the comparison of participants above and below the median cadmium level. Arsenic exposure and metabolism were consistently associated with both epigenetic markers in cross-sectional and prospective analyses. The positive correlation of 5-mC and 5-hmC levels was confirmed in an independent study population. Conclusions: Our findings support that both epigenetic measures are related at the population level. The consistent trends in the associations between these two epigenetic modifications and the characteristics evaluated, especially arsenic exposure and metabolism, suggest the need for understanding which of the two measures is a better biomarker for environmental epigenetic effects in future large-scale epidemiologic studies. Citation: Tellez-Plaza M, Tang WY, Shang Y, Umans JG, Francesconi KA, Goessler W, Ledesma M, Leon M, Laclaustra M, Pollak J, Guallar E, Cole SA, Fallin MD, Navas-Acien A. 2014. Association of global DNA methylation and global DNA hydroxymethylation with metals and other exposures in human blood DNA samples. Environ Health Perspect 122:946–954; http://dx.doi.org/10.1289/ehp.1306674 PMID:24769358

Optimization of techniques for multiple platform testing in small, precious samples such as human chorionic villus sampling.

PubMed

Pisarska, Margareta D; Akhlaghpour, Marzieh; Lee, Bora; Barlow, Gillian M; Xu, Ning; Wang, Erica T; Mackey, Aaron J; Farber, Charles R; Rich, Stephen S; Rotter, Jerome I; Chen, Yii-der I; Goodarzi, Mark O; Guller, Seth; Williams, John

2016-11-01

Multiple testing to understand global changes in gene expression based on genetic and epigenetic modifications is evolving. Chorionic villi, obtained for prenatal testing, is limited, but can be used to understand ongoing human pregnancies. However, optimal storage, processing and utilization of CVS for multiple platform testing have not been established. Leftover CVS samples were flash-frozen or preserved in RNAlater. Modifications to standard isolation kits were performed to isolate quality DNA and RNA from samples as small as 2-5 mg. RNAlater samples had significantly higher RNA yields and quality and were successfully used in microarray and RNA-sequencing (RNA-seq). RNA-seq libraries generated using 200 versus 800-ng RNA showed similar biological coefficients of variation. RNAlater samples had lower DNA yields and quality, which improved by heating the elution buffer to 70 °C. Purification of DNA was not necessary for bisulfite-conversion and genome-wide methylation profiling. CVS cells were propagated and continue to express genes found in freshly isolated chorionic villi. CVS samples preserved in RNAlater are superior. Our optimized techniques provide specimens for genetic, epigenetic and gene expression studies from a single small sample which can be used to develop diagnostics and treatments using a systems biology approach in the prenatal period. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

Quality of human spermatozoa: relationship between high-magnification sperm morphology and DNA integrity.

PubMed

Maettner, R; Sterzik, K; Isachenko, V; Strehler, E; Rahimi, G; Alabart, J L; Sánchez, R; Mallmann, P; Isachenko, E

2014-06-01

The aim of this work is to establish the relationship between the morphology of Intracytoplasmic Morphologically Selected Sperm Injection (IMSI)-selected spermatozoa and their DNA integrity. The 45 ejaculates were randomly distributed into three treatment groups: normozoospermic, oligoasthenozoospermic and oligoasthenotheratozoospermic samples. The evaluation of DNA integrity was performed using the sperm chromatin dispersion test. It was established that DNA integrity of spermatozoa is strongly dependent on ejaculate quality (P < 0.05). The count of spermatozoa with nonfragmented DNA in normozoospermic samples was high and independent from IMSI-morphological classes (Class 1 versus Class 3, respectively) (P > 0.1). With decreased ejaculate quality, the percentage of spermatozoa with nonfragmented DNA decreased significantly (P < 0.05) independent from morphological class. Nevertheless, the rate of IMSI-selected spermatozoa with fragmented DNA within of Class 1 in normozoospermic (Group 1), in oligoasthenozoospermic (Group 2) and in oligoasthenotheratozoospermic (Group 3) samples was 21.1%, 31.8% and 54.1%, respectively. In conclusion, there is a direct relationship between morphological parameters of spermatozoa and their DNA integrity. However, the IMSI technique alone is not enough for the selection of spermatozoa with intact nuclei. © 2013 Blackwell Verlag GmbH.

Long-Term Follow-up of HPV Infection Using Urine and Cervical Quantitative HPV DNA Testing

PubMed Central

Vorsters, Alex; Van Keer, Severien; Biesmans, Samantha; Hens, Annick; De Coster, Ilse; Goossens, Herman; Ieven, Margareta; Van Damme, Pierre

2016-01-01

The link between infection with high-risk human papillomavirus (hrHPV) and cervical cancer has been clearly demonstrated. Virological end-points showing the absence of persistent HPV infection are now accepted as a way of monitoring the impact of prophylactic vaccination programs and therapeutic vaccine trials. This study investigated the use of urine samples, which can be collected by self-sampling at home, instead of cervical samples for follow-up of an HPV intervention trial. Eighteen initially HPV DNA-positive women participating in an HPV therapeutic vaccine trial were monitored during a three-year follow-up period. A total of 172 urine samples and 85 cervical samples were collected. We obtained a paired urine sample for each of the 85 cervical samples by recovering urine samples from six monthly gynaecological examinations. We performed a small pilot study in which the participating women used a urine collection device at home and returned their urine sample to the laboratory by mail. All samples were analyzed using quantitative real-time HPV DNA PCR. A good association (κ value of 0.65) was found between the presence of HPV DNA in urine and a subsequent cervical sample. Comparisons of the number of HPV DNA copies in urine and paired cervical samples revealed a significant Spearman rho of 0.676. This correlation was superior in women with severe lesions. The HPV DNA results of the small pilot study based on self-collected urine samples at home are consistent with previous and subsequent urine and/or cervical results. We demonstrated that urine sampling may be a valid alternative to cervical samples for the follow-up of HPV intervention trials or programs. The potential clinical value of urine viral load monitoring should be further investigated. PMID:27196899

Long-Term Follow-up of HPV Infection Using Urine and Cervical Quantitative HPV DNA Testing.

PubMed

Vorsters, Alex; Van Keer, Severien; Biesmans, Samantha; Hens, Annick; De Coster, Ilse; Goossens, Herman; Ieven, Margareta; Van Damme, Pierre

2016-05-17

The link between infection with high-risk human papillomavirus (hrHPV) and cervical cancer has been clearly demonstrated. Virological end-points showing the absence of persistent HPV infection are now accepted as a way of monitoring the impact of prophylactic vaccination programs and therapeutic vaccine trials. This study investigated the use of urine samples, which can be collected by self-sampling at home, instead of cervical samples for follow-up of an HPV intervention trial. Eighteen initially HPV DNA-positive women participating in an HPV therapeutic vaccine trial were monitored during a three-year follow-up period. A total of 172 urine samples and 85 cervical samples were collected. We obtained a paired urine sample for each of the 85 cervical samples by recovering urine samples from six monthly gynaecological examinations. We performed a small pilot study in which the participating women used a urine collection device at home and returned their urine sample to the laboratory by mail. All samples were analyzed using quantitative real-time HPV DNA PCR. A good association (κ value of 0.65) was found between the presence of HPV DNA in urine and a subsequent cervical sample. Comparisons of the number of HPV DNA copies in urine and paired cervical samples revealed a significant Spearman rho of 0.676. This correlation was superior in women with severe lesions. The HPV DNA results of the small pilot study based on self-collected urine samples at home are consistent with previous and subsequent urine and/or cervical results. We demonstrated that urine sampling may be a valid alternative to cervical samples for the follow-up of HPV intervention trials or programs. The potential clinical value of urine viral load monitoring should be further investigated.

Epigenetic modulations in early endothelial cells and DNA hypermethylation in human skin after sulfur mustard exposure.

PubMed

Steinritz, Dirk; Schmidt, Annette; Balszuweit, Frank; Thiermann, Horst; Simons, Thilo; Striepling, Enno; Bölck, Birgit; Bloch, Wilhelm

2016-02-26

Victims that were exposed to the chemical warfare agent sulfur mustard (SM) suffer from chronic dermal and ocular lesions, severe pulmonary problems and cancer development. It has been proposed that epigenetic perturbations might be involved in that process but this has not been investigated so far. In this study, we investigated epigenetic modulations in vitro using early endothelial cells (EEC) that were exposed to different SM concentrations (0.5, 1.0, 23.5 and 50μM). A comprehensive analysis of 78 genes related to epigenetic pathways (i.e., DNA-methylation and post-translational histone modifications) was performed. Moreover, we analyzed global DNA methylation in vitro in EEC after SM exposure as a maker for epigenetic modulations and in vivo using human skin samples that were obtained from a patient 1 year after an accidently exposure to pure SM. SM exposure resulted in a complex regulation pattern of epigenetic modulators which was accompanied by a global increase of DNA methylation in vitro. Examination of the SM exposed human skin samples also revealed a significant increase of global DNA methylation in vivo, underlining the biological relevance of our findings. Thus, we demonstrated for the first time that SM affects epigenetic pathways and causes epigenetic modulations both in vivo and in vitro. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes.

PubMed

Masuyama, Kotoka; Shojo, Hideki; Nakanishi, Hiroaki; Inokuchi, Shota; Adachi, Noboru

2017-01-01

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted "bidirectional analysis," which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples.

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes

PubMed Central

Masuyama, Kotoka; Shojo, Hideki; Nakanishi, Hiroaki; Inokuchi, Shota; Adachi, Noboru

2017-01-01

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted “bidirectional analysis,” which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples. PMID:28052096

Company profile: Complete Genomics Inc.

PubMed

Reid, Clifford

2011-02-01

Complete Genomics Inc. is a life sciences company that focuses on complete human genome sequencing. It is taking a completely different approach to DNA sequencing than other companies in the industry. Rather than building a general-purpose platform for sequencing all organisms and all applications, it has focused on a single application - complete human genome sequencing. The company's Complete Genomics Analysis Platform (CGA™ Platform) comprises an integrated package of biochemistry, instrumentation and software that sequences human genomes at the highest quality, lowest cost and largest scale available. Complete Genomics offers a turnkey service that enables customers to outsource their human genome sequencing to the company's genome sequencing center in Mountain View, CA, USA. Customers send in their DNA samples, the company does all the library preparation, DNA sequencing, assembly and variant analysis, and customers receive research-ready data that they can use for biological discovery.

Filter forensics: microbiota recovery from residential HVAC filters.

PubMed

Maestre, Juan P; Jennings, Wiley; Wylie, Dennis; Horner, Sharon D; Siegel, Jeffrey; Kinney, Kerry A

2018-01-30

Establishing reliable methods for assessing the microbiome within the built environment is critical for understanding the impact of biological exposures on human health. High-throughput DNA sequencing of dust samples provides valuable insights into the microbiome present in human-occupied spaces. However, the effect that different sampling methods have on the microbial community recovered from dust samples is not well understood across sample types. Heating, ventilation, and air conditioning (HVAC) filters hold promise as long-term, spatially integrated, high volume samplers to characterize the airborne microbiome in homes and other climate-controlled spaces. In this study, the effect that dust recovery method (i.e., cut and elution, swabbing, or vacuuming) has on the microbial community structure, membership, and repeatability inferred by Illumina sequencing was evaluated. The results indicate that vacuum samples captured higher quantities of total, bacterial, and fungal DNA than swab or cut samples. Repeated swab and vacuum samples collected from the same filter were less variable than cut samples with respect to both quantitative DNA recovery and bacterial community structure. Vacuum samples captured substantially greater bacterial diversity than the other methods, whereas fungal diversity was similar across all three methods. Vacuum and swab samples of HVAC filter dust were repeatable and generally superior to cut samples. Nevertheless, the contribution of environmental and human sources to the bacterial and fungal communities recovered via each sampling method was generally consistent across the methods investigated. Dust recovery methodologies have been shown to affect the recovery, repeatability, structure, and membership of microbial communities recovered from dust samples in the built environment. The results of this study are directly applicable to indoor microbiota studies utilizing the filter forensics approach. More broadly, this study provides a better understanding of the microbial community variability attributable to sampling methodology and helps inform interpretation of data collected from other types of dust samples collected from indoor environments.

Molecular Targeting of Prostate Cancer During Androgen Ablation: Inhibition of CHES1/FOXN3

DTIC Science & Technology

2013-05-01

the DNA sequences (~25^6 reads/sample) were mapped to the human genome reference sequence (hg19...tumor the AR has a genomic abnormality, placing the novel sequence 3’ of the transcriptional start site. However, it is unclear if a genomic alteration...exon/intron organization of the CHES1 gene was determined by BLAST analysis of the human genome using the 1,473-bp CHES1 cDNA sequence

Pigment phenotype and biogeographical ancestry from ancient skeletal remains: inferences from multiplexed autosomal SNP analysis.

PubMed

Bouakaze, Caroline; Keyser, Christine; Crubézy, Eric; Montagnon, Daniel; Ludes, Bertrand

2009-07-01

In the present study, a multiplexed genotyping assay for ten single nucleotide polymorphisms (SNPs) located within six pigmentation candidate genes was developed on modern biological samples and applied to DNA retrieved from 25 archeological human remains from southern central Siberia dating from the Bronze and Iron Ages. SNP genotyping was successful for the majority of ancient samples and revealed that most probably had typical European pigment features, i.e., blue or green eye color, light hair color and skin type, and were likely of European individual ancestry. To our knowledge, this study reports for the first time the multiplexed typing of autosomal SNPs on aged and degraded DNA. By providing valuable information on pigment traits of an individual and allowing individual biogeographical ancestry estimation, autosomal SNP typing can improve ancient DNA studies and aid human identification in some forensic casework situations when used to complement conventional molecular markers.

"Would you accept having your DNA profile inserted in the National Forensic DNA database? Why?" Results of a questionnaire applied in Portugal.

PubMed

Machado, Helena; Silva, Susana

2014-01-01

The creation and expansion of forensic DNA databases might involve potential threats to the protection of a range of human rights. At the same time, such databases have social benefits. Based on data collected through an online questionnaire applied to 628 individuals in Portugal, this paper aims to analyze the citizens' willingness to donate voluntarily a sample for profiling and inclusion in the National Forensic DNA Database and the views underpinning such a decision. Nearly one-quarter of the respondents would indicate 'no', and this negative response increased significantly with age and education. The overriding willingness to accept the inclusion of the individual genetic profile indicates an acknowledgement of the investigative potential of forensic DNA technologies and a relegation of civil liberties and human rights to the background, owing to the perceived benefits of protecting both society and the individual from crime. This rationale is mostly expressed by the idea that all citizens should contribute to the expansion of the National Forensic DNA Database for reasons that range from the more abstract assumption that donating a sample for profiling would be helpful in fighting crime to the more concrete suggestion that everyone (criminals and non-criminals) should be in the database. The concerns with the risks of accepting the donation of a sample for genetic profiling and inclusion in the National Forensic DNA Database are mostly related to lack of control and insufficient or unclear regulations concerning safeguarding individuals' data and supervising the access and uses of genetic data. By providing an empirically-grounded understanding of the attitudes regarding willingness to donate voluntary a sample for profiling and inclusion in a National Forensic DNA Database, this study also considers the citizens' perceived benefits and risks of operating forensic DNA databases. These collective views might be useful for the formation of international common ethical standards for the development and governance of DNA databases in a framework in which the citizens' perspectives are taken into consideration. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Increased levels of mitochondrial DNA copy number in patients with vitiligo.

PubMed

Vaseghi, H; Houshmand, M; Jadali, Z

2017-10-01

Oxidative stress is known to be involved in the pathogenesis of autoimmune diseases such as vitiligo. Evidence suggests that the human mitochondrial DNA copy number (mtDNAcn) is vulnerable to damage mediated by oxidative stress. The purpose of this study was to examine and compare peripheral blood mtDNAcn and oxidative DNA damage byproducts (8-hydroxy-2-deoxyguanosine; 8-OHdG) in patients with vitiligo and healthy controls (HCs). The relative mtDNAcn and the oxidative damage (formation of 8-OHdG in mtDNA) of each sample were determined by real-time quantitative PCR. Blood samples were obtained from 56 patients with vitiligo and 46 HCs. The mean mtDNAcn and the degree of mtDNA damage were higher in patients with vitiligo than in HCs. These data suggest that increase in mtDNAcn and oxidative DNA damage may be involved in the pathogenesis of vitiligo. © 2017 British Association of Dermatologists.

Predominant bacterial species in subgingival plaque in dogs.

PubMed

Dahlén, G; Charalampakis, G; Abrahamsson, I; Bengtsson, L; Falsen, E

2012-06-01

The dog has been used extensively for experimental and microbiological studies on periodontitis and peri-implantitis without detailed knowledge about the predominant flora of the subgingival plaque. This study was designed to evaluate the predominant cultivable bacterial species in dogs and compare them phenotypically and genotypically with corresponding human species. Four subgingival samples were taken from two upper premolars in each of six Labrador retrievers. The samples from each dog were processed for anaerobic culture. From the samples of each dog, the five or six predominating bacteria based on colony morphology were selected and pure cultured. Each of the strains was characterized by Gram stain, anaerobic/aerobic growth and API-ZYM test. Eighteen strains showing clear-cut phenotypic differences were further classified based on DNA sequencing technology. Cross-reactions of DNA probes from human and dog strains were also tested against a panel of both human and dog bacterial species. Thirty-one strains in the dogs were isolated and characterized. They represented 21 different species, of which six belonged to the genus Porphyromonas. No species was found consistently in the predominant flora of all six dogs. Porphyromonas crevioricanis and Fusobacterium canifelinum were the two most prevalent species in predominant flora in dogs. DNA probes from human and dog species cross-reacted to some extent with related strains from humans and dogs; however, distinct exceptions were found. The predominant cultural subgingival flora in dogs shows great similarities with the subgingival bacteria from humans at the genus level, but distinct differences at the species level; however, a genetic relatedness could be disclosed for most strains investigated. © 2011 John Wiley & Sons A/S.

Determinants of Viral Oncogene E6-E7 mRNA Overexpression in a Population-Based Large Sample of Women Infected by High-Risk Human Papillomavirus Types

PubMed Central

Bisanzi, Simonetta; Allia, Elena; Mongia, Alessandra; Carozzi, Francesca; Gillio-Tos, Anna; De Marco, Laura; Ronco, Guglielmo; Gustinucci, Daniela; Del Mistro, Annarosa; Frayle, Helena; Iossa, Anna; Fantacci, Giulia; Pompeo, Giampaolo; Cesarini, Elena; Bulletti, Simonetta; Passamonti, Basilio; Rizzi, Martina; Penon, Maria Gabriella; Barca, Alessandra; Benevolo, Maria

2017-01-01

ABSTRACT Cervical cancer screening by human papillomavirus (HPV) DNA testing with cytology triage is more effective than cytology testing. Compared to cytology, the HPV DNA test's higher sensitivity, which allows better protection with longer intervals, makes it necessary to triage the women with a positive result to compensate its lower specificity. We are conducting a large randomized clinical trial (New Technologies for Cervical Cancer 2 [NTCC2]) within organized population-based screening programs in Italy using HPV DNA as the primary screening test to evaluate, by the Aptima HPV assay (Hologic), the use of HPV E6-E7 mRNA in a triage test in comparison to cytology. By the end of June 2016, data were available for 35,877 of 38,535 enrolled women, 2,651 (7.4%) of whom were HPV DNA positive. Among the samples obtained, 2,453 samples were tested also by Aptima, and 1,649 (67.2%) gave a positive result. The proportion of mRNA positivity was slightly higher among samples tested for HPV DNA by the Cobas 4800 HPV assay (Roche) than by the Hybrid Capture 2 (HC2) assay (Qiagen). In our setting, the observed E6-E7 mRNA positivity rate, if used as a triage test, would bring a rate of immediate referral to colposcopy of about 4 to 5%. This value is higher than that observed with cytology triage for both immediate and delayed referrals to colposcopy. By showing only a very high sensitivity and thus allowing a longer interval for HPV DNA-positive/HPV mRNA-negative women, a triage by this test might be more efficient than by cytology. PMID:28100595

Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing.

PubMed

Thoendel, Matthew; Jeraldo, Patricio R; Greenwood-Quaintance, Kerryl E; Yao, Janet Z; Chia, Nicholas; Hanssen, Arlen D; Abdel, Matthew P; Patel, Robin

2016-08-01

Metagenomic whole genome sequencing for detection of pathogens in clinical samples is an exciting new area for discovery and clinical testing. A major barrier to this approach is the overwhelming ratio of human to pathogen DNA in samples with low pathogen abundance, which is typical of most clinical specimens. Microbial DNA enrichment methods offer the potential to relieve this limitation by improving this ratio. Two commercially available enrichment kits, the NEBNext Microbiome DNA Enrichment Kit and the Molzym MolYsis Basic kit, were tested for their ability to enrich for microbial DNA from resected arthroplasty component sonicate fluids from prosthetic joint infections or uninfected sonicate fluids spiked with Staphylococcus aureus. Using spiked uninfected sonicate fluid there was a 6-fold enrichment of bacterial DNA with the NEBNext kit and 76-fold enrichment with the MolYsis kit. Metagenomic whole genome sequencing of sonicate fluid revealed 13- to 85-fold enrichment of bacterial DNA using the NEBNext enrichment kit. The MolYsis approach achieved 481- to 9580-fold enrichment, resulting in 7 to 59% of sequencing reads being from the pathogens known to be present in the samples. These results demonstrate the usefulness of these tools when testing clinical samples with low microbial burden using next generation sequencing. Copyright © 2016 Elsevier B.V. All rights reserved.

'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples.

PubMed

Eichmann, Cordula; Parson, Walther

2008-09-01

The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation.

Collecting, archiving and processing DNA from wildlife samples using FTA® databasing paper

PubMed Central

Smith, LM; Burgoyne, LA

2004-01-01

Background Methods involving the analysis of nucleic acids have become widespread in the fields of traditional biology and ecology, however the storage and transport of samples collected in the field to the laboratory in such a manner to allow purification of intact nucleic acids can prove problematical. Results FTA® databasing paper is widely used in human forensic analysis for the storage of biological samples and for purification of nucleic acids. The possible uses of FTA® databasing paper in the purification of DNA from samples of wildlife origin were examined, with particular reference to problems expected due to the nature of samples of wildlife origin. The processing of blood and tissue samples, the possibility of excess DNA in blood samples due to nucleated erythrocytes, and the analysis of degraded samples were all examined, as was the question of long term storage of blood samples on FTA® paper. Examples of the end use of the purified DNA are given for all protocols and the rationale behind the processing procedures is also explained to allow the end user to adjust the protocols as required. Conclusions FTA® paper is eminently suitable for collection of, and purification of nucleic acids from, biological samples from a wide range of wildlife species. This technology makes the collection and storage of such samples much simpler. PMID:15072582

Isolation of human genomic DNA for genetic analysis from premature neonates: a comparison between newborn dried blood spots, whole blood and umbilical cord tissue

PubMed Central

2013-01-01

Background Genotyping requires biological sample collection that must be reliable, convenient and acceptable for patients and clinicians. Finding the most optimal procedure of sample collection for premature neonates who have a very limited blood volume is a particular challenge. The aim of the current study was to evaluate the use of umbilical cord (UC) tissue and newborn dried blood spot (DBS)-extracted genomic DNA (gDNA) as an alternative to venous blood-derived gDNA from premature neonates for molecular genetic analysis. All samples were obtained from premature newborn infants between 24-32 weeks of gestation. Paired blood and UC samples were collected from 31 study participants. gDNA was extracted from ethylenediaminetetraacetic acid (EDTA) anticoagulant-treated blood samples (~500 μl) and newborn DBSs (n = 723) using QIAamp DNA Micro kit (Qiagen Ltd., Crawley, UK); and from UC using Qiagen DNAeasy Blood and Tissue kit (Qiagen Ltd., Crawley, UK). gDNA was quantified and purity confirmed by measuring the A260:A280 ratio. PCR amplification and pyrosequencing was carried out to determine suitability of the gDNA for molecular genetic analysis. Minor allele frequency of two unrelated single nucleotide polymorphisms (SNPs) was calculated using the entire cohort. Results Both whole blood samples and UC tissue provided good quality and yield of gDNA, which was considerably less from newborn DBS. The gDNA purity was also reduced after 3 years of storage of the newborn DBS. PCR amplification of three unrelated genes resulted in clear products in all whole blood and UC samples and 86%-100% of newborn DBS. Genotyping using pyrosequencing showed 100% concordance in the paired UC and whole blood samples. Minor allele frequencies of the two SNPs indicated that no maternal gDNA contamination occurred in the genotyping of the UC samples. Conclusions gDNAs from all three sources are suitable for standard PCR and pyrosequencing assays. Given that UC provide good quality and quantity gDNA with 100% concordance in the genetic analysis with whole blood, it can replace blood sampling from premature infants. This is likely to reduce the stress and potential side effects associated with invasive sample collection and thus, greatly facilitate participant recruitment for genetic studies. PMID:24168095

[Effects of hepatitis B virus on human semen parameters and sperm DNA integrity].

PubMed

Liu, Hao; Geng, Chun-Hui; Wang, Wei; Xiao, Ke-Lin; Xiong, Li-Kuan; Huang, Yong-Xiang; Yang, Xiao-Ling; Li, Jin

2013-10-01

To investigate the effects of hepatitis B virus (HBV) in semen on human semen parameters and sperm DNA integrity. We detected HBV DNA in the semen samples of 153 HBsAg-seropositive patients by real-time fluorescence quantitative PCR and calculated the sperm nuclear DNA fragmentation index (DFI) by sperm chromatin dispersion (SCD) assay. We compared the semen parameters between the HBV DNA-positive group (A, n = 43) and HBV DNA-negative group (B, n = 110) and analyzed the correlation of sperm DFI with the number of HBV DNA copies in the semen. HBV DNA was detected in 43 (28.1%) of the 153 semen samples. No statistically significant differences were observed in age, semen volume and sperm concentration between groups A and B (P >0.05). Compared with group B, group A showed significantly decreased sperm viability ([58.0 +/- 18.8]% vs [51.4 +/-17.1]%, P<0.05), progressively motile sperm ([29.6 +/- 13.3]% vs [24.5 +/- 10.1]%, P<0.05), average straight-line velocity ([23.7 +/- 4.0] microm/s vs [19.9 +/- 4.5 ] microm/s, P<0.01) and average path velocity ([26.5 +/- 7.0] microm/s vs [23.4 +/- 5.3] microm/s, P<0.01), but remarkably decreased sperm DFI ([19.3 +/- 8.0]% vs [24.2 +/- 9.4]%, P<0.01). The number of HBV DNA copies in semen exhibited a significant positive correlation with sperm DFI (r = 0.819, P < 0.01). HBV DNA in semen is not significantly associated with the number of sperm, but may affect sperm viability, velocity and DFI. There is a load-effect relationship between the number of HBV DNA copies in semen and sperm nuclear DNA integrity.

Biobanking human endometrial tissue and blood specimens: standard operating procedure and importance to reproductive biology research and diagnostic development.

PubMed

Sheldon, Elizabeth; Vo, Kim Chi; McIntire, Ramsey A; Aghajanova, Lusine; Zelenko, Zara; Irwin, Juan C; Giudice, Linda C

2011-05-01

To develop a standard operating procedure (SOP) for collection, transport, storage of human endometrial tissue and blood samples, subject and specimen annotation, and establishing sample priorities. The SOP synthesizes sound scientific procedures, the literature on ischemia research, sample collection and gene expression profiling, good laboratory practices, and the authors' experience of workflow and sample quality. The National Institutes of Health, University of California, San Francisco, Human Endometrial Tissue and DNA Bank. Women undergoing endometrial biopsy or hysterectomy for nonmalignant indications. Collecting, processing, storing, distributing endometrial tissue and blood samples under approved institutional review board protocols and written informed consent from participating subjects. Standard operating procedure. The SOP addresses rigorous and consistent subject annotation, specimen processing and characterization, strict regulatory compliance, and a reference for researchers to track collection and storage times that may influence their research. The comprehensive and systematic approach to the procurement of human blood and endometrial tissue in this SOP ensures the high quality, reliability, and scientific usefulness of biospecimens made available to investigators by the National Institutes of Health, University of California, San Francisco, Human Endometrial Tissue and DNA Bank. The detail and perspective in this SOP also provides a blueprint for implementation of similar collection programs at other institutions. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Association Between Schizophrenia and DNA Demethylase Activity in Human Peripheral Blood Mononuclear Cells.

PubMed

Zhang, Lili; Pang, Bo; Zhang, Wenbin; Bai, Wei; Yu, Weiying; Li, Yuanyuan; Hua, Wanqing; Li, Wenjun; Kou, Changgui

2018-06-01

DNA demethylase is a crucial enzyme in the epigenetic modification and regulation mechanisms of gene transcription. Based on previous assertions that the pathophysiology of schizophrenia is associated with epigenetics, we aimed to explore whether DNA demethylase activity might be related to schizophrenia in northeast China. We recruited 25 patients with first-episode schizophrenia and 29 normal controls from a northeast Chinese Han population. The diagnostic criteria of schizophrenia were determined according to diseases and related health problems, the tenth revision (ICD-10), and criteria of mental disorders, the third revised edition (CCMD3). DNA demethylase activity in human peripheral blood mononuclear cells (PBMCs) was measured using a DNA demethylase activity colorimetric assay ultra kit. Using Student's t-test, activation of DNA demethylase and its activity were higher in schizophrenia patients compared to healthy individuals (p < 0.001). Furthermore, the level of DNA demethylase activity in male and female subjects with schizophrenia significantly increased (all p < 0.05). Our data showed that DNA demethylase might play a role in the pathophysiology of schizophrenia, and individuals with higher DNA demethylase activity were susceptible to schizophrenia in a northeast Chinese Han population. To the best of our knowledge, this is the first time directly measured human blood samples to examine the association between first-episode schizophrenia patients and DNA demethylase activity, which will provide new insight to explore the effect on the mechanism of schizophrenia.

The effect of common imaging and hot water maceration on DNA recovery from skeletal remains.

PubMed

Frank, Emilie M; Mundorff, Amy Z; Davoren, Jon M

2015-12-01

Identifying human remains often begins with cleaning and imaging the material. Hot water maceration is used to remove adherent soft tissue from bone and radiographs are taken to better visualize osseous details. Heat and radiation are known to have harmful effects on DNA, but their ability to degrade DNA when used for cleaning and imaging has not been well studied. To better understand their individual and combined effects on the recoverability of DNA from bone, skeletal samples were subjected to (1) hot water maceration (62 °C for 45 min); (2) CT scanning (0.6mm slices, 120 kV, 10.4s); (3) X-ray (50 kVp, 150 mA, 0.03 s, 40 in); and (4) all 3 treatments combined. Forty-eight DNA samples were extracted, quantified and amplified with the AmpFLSTR(®) Identifiler(®) system. Nearly all of the processed samples had reduced RFU values relative to the unprocessed samples, indicating some amount of genetic loss. This loss did not always translate into loss of profile completeness, since only a few samples had a reduction in the number of loci detected after processing. DNA yields were not significantly reduced by any one of the processing methods, however the results indicate that the damaging effects are additive. It is possible that processing may reduce a bone's DNA reservoir and as more procedures are preformed, the pool of available genetic information might be diminished. Many intrinsic and extrinsic factors can affect the recoverability of DNA from bone. Collecting a DNA sample prior to processing avoids the negative effects from hot water maceration and radiological imaging. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Freezer anthropology: new uses for old blood.

PubMed Central

Merriwether, D A

1999-01-01

Archived blood fractions (plasma, settled red cells, white cells) have proved to be a rich and valuable source of DNA for human genetic studies. Large numbers of such samples were collected between 1960 and the present for protein and blood group studies, many of which are languishing in freezers or have already been discarded. More are discarded each year because the usefulness of these samples is not widely understood. Data from DNA derived from 10-35-year-old blood samples have been used to address the peopling of the New World and of the Pacific. Mitochondrial DNA haplotypes from studies using this source DNA support a single wave of migration into the New World (or a single source population for the New World), and that Mongolia was the likely source of the founding population. Data from Melanesia have shown that Polynesians are recent immigrants into the Pacific and did not arise from Melanesia. PMID:10091252

In vivo investigations of the effect of short- and long-term recombinant growth hormone treatment on DNA-methylation in humans.

PubMed

Kolarova, Julia; Ammerpohl, Ole; Gutwein, Jana; Welzel, Maik; Baus, Inka; Riepe, Felix G; Eggermann, Thomas; Caliebe, Almuth; Holterhus, Paul-Martin; Siebert, Reiner; Bens, Susanne

2015-01-01

Treatment with recombinant human growth hormone (rhGH) has been consistently reported to induce transcriptional changes in various human tissues including peripheral blood. For other hormones it has been shown that the induction of such transcriptional effects is conferred or at least accompanied by DNA-methylation changes. To analyse effects of short term rhGH treatment on the DNA-methylome we investigated a total of 24 patients at baseline and after 4-day rhGH stimulation. We performed array-based DNA-methylation profiling of paired peripheral blood mononuclear cell samples followed by targeted validation using bisulfite pyrosequencing. Unsupervised analysis of DNA-methylation in this short-term treated cohort revealed clustering according to individuals rather than treatment. Supervised analysis identified 239 CpGs as significantly differentially methylated between baseline and rhGH-stimulated samples (p<0.0001, unadjusted paired t-test), which nevertheless did not retain significance after adjustment for multiple testing. An individualized evaluation strategy led to the identification of 2350 CpG and 3 CpH sites showing methylation differences of at least 10% in more than 2 of the 24 analyzed sample pairs. To investigate the long term effects of rhGH treatment on the DNA-methylome, we analyzed peripheral blood cells from an independent cohort of 36 rhGH treated children born small for gestational age (SGA) as compared to 18 untreated controls. Median treatment interval was 33 months. In line with the groupwise comparison in the short-term treated cohort no differentially methylated targets reached the level of significance in the long-term treated cohort. We identified marked intra-individual responses of DNA-methylation to short-term rhGH treatment. These responses seem to be predominately associated with immunologic functions and show considerable inter-individual heterogeneity. The latter is likely the cause for the lack of a rhGH induced homogeneous DNA-methylation signature after short- and long-term treatment, which nevertheless is well in line with generally assumed safety of rhGH treatment.

Detection of Cytosine methylation in ancient DNA from five native american populations using bisulfite sequencing.

PubMed

Smith, Rick W A; Monroe, Cara; Bolnick, Deborah A

2015-01-01

While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches.

Comparative evaluation of environmental contamination and DNA damage induced by electronic-waste in Nigeria and China.

PubMed

Alabi, Okunola A; Bakare, Adekunle A; Xu, Xijin; Li, Bin; Zhang, Yuling; Huo, Xia

2012-04-15

In the last decade, China and Nigeria have been prime destinations for the world's e-waste disposal leading to serious environmental contamination. We carried out a comparative study of the level of contamination using soils and plants from e-waste dumping and processing sites in both countries. Levels of polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs) were analyzed using gas chromatography/spectrophotometry and heavy metals using atomic absorption spectrophotometry. DNA damage was assayed in human peripheral blood lymphocytes using an alkaline comet assay. Soils and plants were highly contaminated with toxic PAHs, PCBs, PBDEs, and heavy metals in both countries. Soil samples from China and plant samples from Nigeria were more contaminated. There was a positive correlation between the concentrations of organics and heavy metals in plant samples and the surrounding soils. In human lymphocytes, all tested samples induced significant (p<0.05) concentration-dependent increases in DNA damage compared with the negative control. These findings suggest that e-waste components/constituents can accumulate, in soil and surrounding vegetation, to toxic and genotoxic levels that could induce adverse health effects in exposed individuals. Copyright © 2012 Elsevier B.V. All rights reserved.

Pediatric epstein-barr virus carriers with or without tonsillar enlargement may substantially contribute to spreading of the virus.

PubMed

Hug, Martina; Dorner, Marcus; Fröhlich, Franziska Zucol; Gysin, Claudine; Neuhaus, Diego; Nadal, David; Berger, Christoph

2010-10-15

Human-to-human transmission of the persistent infection establishing Epstein-Barr virus (EBV) occurs via saliva. Tonsils act as important portal of entry and exit of EBV. The contagiousness of pediatric EBV carriers and the role played by tonsillar enlargement (TE) are not known. We compared EBV shedding in mouthwash samples from pediatric EBV carriers with or without TE to that in mouthwash samples from pediatric patients with infectious mononucleosis (IM), the symptomatic form of primary infection if delayed after the age of 5 years. EBV DNA was quantified by polymerase chain reaction, and contagiousness was assessed using the cord lymphocyte transformation assay. EBV carriers with TE shed EBV DNA at an almost similar frequency (although in lower amounts) as pediatric patients with acute IM but more frequently (P <.001) and in higher amounts (P = .038) than EBV carriers without TE. EBV DNA levels in mouthwash samples from EBV carriers with TE mirrored levels in tonsils and gradually declined after tonsillectomy. Almost half of the mouthwash samples from pediatric EBV carriers contained infectious EBV. Pediatric EBV carriers--in particular, those with TE-may considerably contribute to the spreading of EBV in industrialized countries.

Is High Resolution Melting Analysis (HRMA) Accurate for Detection of Human Disease-Associated Mutations? A Meta Analysis

PubMed Central

Ma, Feng-Li; Jiang, Bo; Song, Xiao-Xiao; Xu, An-Gao

2011-01-01

Background High Resolution Melting Analysis (HRMA) is becoming the preferred method for mutation detection. However, its accuracy in the individual clinical diagnostic setting is variable. To assess the diagnostic accuracy of HRMA for human mutations in comparison to DNA sequencing in different routine clinical settings, we have conducted a meta-analysis of published reports. Methodology/Principal Findings Out of 195 publications obtained from the initial search criteria, thirty-four studies assessing the accuracy of HRMA were included in the meta-analysis. We found that HRMA was a highly sensitive test for detecting disease-associated mutations in humans. Overall, the summary sensitivity was 97.5% (95% confidence interval (CI): 96.8–98.5; I2 = 27.0%). Subgroup analysis showed even higher sensitivity for non-HR-1 instruments (sensitivity 98.7% (95%CI: 97.7–99.3; I2 = 0.0%)) and an eligible sample size subgroup (sensitivity 99.3% (95%CI: 98.1–99.8; I2 = 0.0%)). HRMA specificity showed considerable heterogeneity between studies. Sensitivity of the techniques was influenced by sample size and instrument type but by not sample source or dye type. Conclusions/Significance These findings show that HRMA is a highly sensitive, simple and low-cost test to detect human disease-associated mutations, especially for samples with mutations of low incidence. The burden on DNA sequencing could be significantly reduced by the implementation of HRMA, but it should be recognized that its sensitivity varies according to the number of samples with/without mutations, and positive results require DNA sequencing for confirmation. PMID:22194806

Quantitation of human immunodeficiency virus type 1 in breast milk.

PubMed

Ghosh, M K; Kuhn, L; West, J; Semrau, K; Decker, D; Thea, D M; Aldrovandi, G M

2003-06-01

The distribution and stability of human immunodeficiency virus type 1 (HIV-1) in breast milk (BM) components remain largely unknown. Inhibitory effects, if any, of BM on HIV RNA and DNA PCR amplification are poorly understood. We have addressed these issues by using virus-spiked BM samples from HIV-negative women. BM samples from HIV-negative women were spiked with HIV-1 virions or cells containing a single integrated copy of HIV DNA (8E5/LAV). After incubation under different experimental conditions, viral RNA was detected by the Roche Amplicor UltraSensitive assay in whole-milk, skim milk, and lipid fractions. We found excellent correlation between HIV-1 input copy and recovery in whole milk (r = 0.965, P < 0.0001), skim milk (r = 0.972, P < 0.0001), and the lipid fraction (r = 0.905, P < 0.001). PCR inhibition was observed in less than 10% of the spiked samples. Similar levels of inhibition were noted in BM samples collected from HIV-infected women. HIV proviral DNA was detected in BM samples using real-time PCR (linear correlation between the threshold cycle versus log DNA copy number, >0.982). The effects of incubation duration and temperature and repeated freeze-thaw cycles on HIV RNA recovery were analyzed. HIV RNA levels were remarkably stable in whole milk after three freeze-thaw cycles and for up to 30 h at room temperature. Our findings improve the understanding of the dynamics of HIV detection in BM and the conditions for BM sample collection, storage, and processing.

A field survey using LAMP assay for detection of Schistosoma mansoni in a low-transmission area of schistosomiasis in Umbuzeiro, Brazil: Assessment in human and snail samples.

PubMed

Gandasegui, Javier; Fernández-Soto, Pedro; Muro, Antonio; Simões Barbosa, Constança; Lopes de Melo, Fabio; Loyo, Rodrigo; de Souza Gomes, Elainne Christine

2018-03-01

In Brazil, schistosomiasis is a parasitic disease of public health relevance, mainly in poor areas where Schistosoma mansoni is the only human species encountered and Biomphalaria straminea is one of the intermediate host snails. A nested-PCR based on a specific mitochondrial S. mansoni minisatellite DNA region has been successfully developed and applied as a reference method in Brazil for S. mansoni detection, mainly in host snails for epidemiological studies. The amplification efficiency of LAMP is known to be higher than PCR. The present work aimed to assess the utility of our previously described SmMIT-LAMP assay for S. mansoni detection in human stool and snail samples in a low-transmission area of schistosomiasis in the municipality of Umbuzeiro, Paraíba State, Northeast Region of Brazil. A total of 427 human stool samples were collected during June-July 2016 in the municipality of Umbuzeiro and an overall prevalence of 3.04% (13/427) resulted positive by duplicate Kato-Katz thick smear. A total of 1,175 snails identified as Biomphalaria straminea were collected from 14 breeding sites along the Paraíba riverbank and distributed in 46 pools. DNA from human stool samples and pooled snails was extracted using the phenol/chloroform method. When performing the SmMIT-LAMP assay a total of 49/162 (30.24%) stool samples resulted positive, including 12/13 (92.31%) that were Kato-Katz positive and 37/149 (24.83%) previously Kato-Katz negative. By nested-PCR, only 1/46 pooled DNA snail samples was positive. By SmMIT-LAMP assay, the same sample also resulted positive and an additional one was positive from a different breeding site. Data of human and snail surveys were used to build risk maps of schistosomiasis incidence using kernel density analysis. This is the first study in which a LAMP assay was evaluated in both human stool and snail samples from a low-transmission schistosomiasis-endemic area. Our SmMIT-LAMP proved to be much more efficient in detection of S. mansoni in comparison to the 'gold standard' Kato-Katz method in human stool samples and the reference molecular nested-PCR in snails. The SmMIT-LAMP has demonstrated to be a useful molecular tool to identify potential foci of transmission in order to build risk maps of schistosomiasis.

Is urbanisation scrambling the genetic structure of human populations? A case study

PubMed Central

Ashrafian-Bonab, Maziar; Handley, Lori Lawson; Balloux, François

2007-01-01

Recent population expansion and increased migration linked to urbanisation are assumed to be eroding the genetic structure of human populations. We investigated change in population structure over three generations by analysing both demographic and mitochondrial DNA (mtDNA) data from a random sample of 2351 men from twenty-two Iranian populations. Potential changes in genetic diversity (θ) and genetic distance (FST) over the last three generations were analysed by assigning mtDNA sequences to populations based on the individual's place of birth or that of their mother or grandmother. Despite the fact that several areas included cities of over one million inhabitants, we detected no change in genetic diversity, and only a small decrease in population structure, except in the capital city (Tehran), which was characterised by massive immigration, increased θ and a large decrease in FST over time. Our results suggest that recent erosion of human population structure might not be as important as previously thought, except in some large conurbations, and this clearly has important implications for future sampling strategies. PMID:17106453

Presence of Borrelia spp. DNA in ticks, but absence of Borrelia spp. and of Leptospira spp. DNA in blood of fever patients in Madagascar.

PubMed

Hagen, Ralf Matthias; Frickmann, Hagen; Ehlers, Julian; Krüger, Andreas; Margos, Gabriele; Hizo-Teufel, Cecilia; Fingerle, Volker; Rakotozandrindrainy, Raphael; Kalckreuth, Vera von; Im, Justin; Pak, Gi Deok; Jeon, Hyon Jin; Rakotondrainiarivelo, Jean Philibert; Heriniaina, Jean Noël; Razafindrabe, Tsiry; Konings, Frank; May, Jürgen; Hogan, Benedikt; Ganzhorn, Jörg; Panzner, Ursula; Schwarz, Norbert Georg; Dekker, Denise; Marks, Florian; Poppert, Sven

2018-01-01

The occurrence of tick-borne relapsing fever and leptospirosis in humans in Madagascar remains unclear despite the presence of their potential vectors and reservoir hosts. We screened 255 Amblyomma variegatum ticks and 148 Rhipicephalus microplus ticks from Zebu cattle in Madagascar for Borrelia-specific DNA. Borrelia spp. DNA was detected in 21 Amblyomma variegatum ticks and 2 Rhipicephalus microplus ticks. One Borrelia found in one Rhipicephalus microplus showed close relationship to Borrelia theileri based on genetic distance and phylogenetic analyses on 16S rRNA and flaB sequences. The borreliae from Amblyomma variegatum could not be identified due to very low quantities of present DNA reflected by high cycle threshold values in real-time-PCR. It is uncertain whether these low numbers of Borrelia spp. are sufficient for transmission of infection from ticks to humans. In order to determine whether spirochaete infections are relevant in humans, blood samples of 1009 patients from the highlands of Madagascar with fever of unknown origin were screened for Borrelia spp. - and in addition for Leptospira spp. - by real-time PCR. No target DNA was detected, indicating a limited relevance of these pathogens for humans in the highlands of Madagascar. Copyright © 2017 Elsevier B.V. All rights reserved.

[Correlation of genomic DNA methylation level with unexplained early spontaneous abortion].

PubMed

Chao, Yuan; Weng, Lidong; Zeng, Rong

2014-10-01

To investigate the correlation of genomic DNA methylation level with unexplained early spontaneous abortion and analyze the role of DNMT1, DNMT3A and DNMT3B. Forty-five villus samples from spontaneous abortion cases (with 33 maternal peripheral blood samples) and 44 villus samples from induced abortion (with 34 maternal peripheral blood samples) were examined with high-pressure liquid chromatography (HPLC) to measure the overall methylation level of the genomic DNA. The expressions of DNMT mRNAs were detected using fluorescence quantitative-PCR in the villus samples from 33 induced abortion cases and 30 spontaneous abortion cases. Genomic DNA methylation level was significantly lower in the villus in spontaneous abortion group than in induced abortion group (P<0.01), but similar in the maternal blood samples between the two groups (P>0.05). The mean mRNA expression levels of DNMT1 and DNMT3A in the villus were significantly lower in spontaneous abortion group than in induced abortion group (P<0.05), but DNMT3B expression showed no significant difference between them (P>0.05). Insufficient genomic DNA methylation in the villus does exist in human early spontaneous abortion, and this insufficiency is probably associated with down-regulated expressions of DNMT1 and DNMT3A.

Fellow travellers: a concordance of colonization patterns between mice and men in the North Atlantic region.

PubMed

Jones, E P; Skirnisson, K; McGovern, T H; Gilbert, M T P; Willerslev, E; Searle, J B

2012-03-19

House mice (Mus musculus) are commensals of humans and therefore their phylogeography can reflect human colonization and settlement patterns. Previous studies have linked the distribution of house mouse mitochondrial (mt) DNA clades to areas formerly occupied by the Norwegian Vikings in Norway and the British Isles. Norwegian Viking activity also extended further westwards in the North Atlantic with the settlement of Iceland, short-lived colonies in Greenland and a fleeting colony in Newfoundland in 1000 AD. Here we investigate whether house mouse mtDNA sequences reflect human history in these other regions as well. House mice samples from Iceland, whether from archaeological Viking Age material or from modern-day specimens, had an identical mtDNA haplotype to the clade previously linked with Norwegian Vikings. From mtDNA and microsatellite data, the modern-day Icelandic mice also share the low genetic diversity shown by their human hosts on Iceland. Viking Age mice from Greenland had an mtDNA haplotype deriving from the Icelandic haplotype, but the modern-day Greenlandic mice belong to an entirely different mtDNA clade. We found no genetic association between modern Newfoundland mice and the Icelandic/ancient Greenlandic mice (no ancient Newfoundland mice were available). The modern day Icelandic and Newfoundland mice belong to the subspecies M. m. domesticus, the Greenlandic mice to M. m. musculus. In the North Atlantic region, human settlement history over a thousand years is reflected remarkably by the mtDNA phylogeny of house mice. In Iceland, the mtDNA data show the arrival and continuity of the house mouse population to the present day, while in Greenland the data suggest the arrival, subsequent extinction and recolonization of house mice--in both places mirroring the history of the European human host populations. If house mice arrived in Newfoundland with the Viking settlers at all, then, like the humans, their presence was also fleeting and left no genetic trace. The continuity of mtDNA haplotype in Iceland over 1000 years illustrates that mtDNA can retain the signature of the ancestral house mouse founders. We also show that, in terms of genetic variability, house mouse populations may also track their host human populations.

Mitochondrial Debris as a Discriminator Between Inflammatory and Infectious Complications of Blast Injuries: The Enemy Within

DTIC Science & Technology

2012-01-01

adult respiratory distress syndrome (ALI/ARDS) occur after fractures in a sporadic entity often termed ‘‘ fat embolism syndrome.’’ Fat embolism ...We then went on to evaluate the role of fracture injuries in mobilizing mtDNA from human tissue trauma. As proposed, we used discarded human samples...to show that long bone fractures (very common in combatants) and their repair by clinical reamed nailing operations mobilized huge amounts of mtDNA

Investigating the Prevalence of Human Papilloma Virus in Squamous Cell Carcinoma of the Larynx and Its Correlation with Disease Prognosis.

PubMed

Atighechi, Saeid; Meybodian, Mojtaba; Dadgarnia, Mohammad Hossein; Baradaranfar, Mohammad Hossein; Behniafard, Nasim

2016-05-01

The human papilloma virus (HPV) can play a role in the development of head and neck squamous cell carcinoma (SCC). Our aim was to assess the prevalence of HPV DNA in SCC of the larynx. The impact of HPV infection on patient survival was also evaluated. This case-control study was performed in 44 patients with SCC of the larynx (case group), while the control group comprised samples obtained from cadavers with no previous history of malignancy. A preliminary pathologic evaluation was performed on all samples in the control group (36 samples) to ensure the absence of dysplasia or malignancy. Polymerase chain reaction (PCR) was used to detect HPV DNA. After completing the treatment protocol, patients were followed to assess the impact of HPV infection on overall survival (OS). PCR evaluation in the case group showed that HPV DNA was successfully isolated from 11 (25%) samples, while only two (5.6%) HPV DNA-positive were obtained from cadavers. According to these results, a significant difference was obtained in the prevalence of HPV DNA and laryngeal SCC between cases and controls (P=0.031). No statistically significant difference was observed in the OS of patients with or without HPV infection in the case group (P=0.235). Based on these results, we suggest that the prevalence of HPV infection is higher in laryngeal SCC subjects compared with healthy individuals. Although a longer OS was seen in HPV-positive patients, survival analysis did not show a significant difference in the comparison of HPV-positive and negative findings in SCC patients.

Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella Species in Human Feces by Using Group-Specific PCR Primers and Denaturing Gradient Gel Electrophoresis

PubMed Central

Walter, Jens; Hertel, Christian; Tannock, Gerald W.; Lis, Claudia M.; Munro, Karen; Hammes, Walter P.

2001-01-01

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects. PMID:11375166

Gold nanoparticles for high-throughput genotyping of long-range haplotypes

NASA Astrophysics Data System (ADS)

Chen, Peng; Pan, Dun; Fan, Chunhai; Chen, Jianhua; Huang, Ke; Wang, Dongfang; Zhang, Honglu; Li, You; Feng, Guoyin; Liang, Peiji; He, Lin; Shi, Yongyong

2011-10-01

Completion of the Human Genome Project and the HapMap Project has led to increasing demands for mapping complex traits in humans to understand the aetiology of diseases. Identifying variations in the DNA sequence, which affect how we develop disease and respond to pathogens and drugs, is important for this purpose, but it is difficult to identify these variations in large sample sets. Here we show that through a combination of capillary sequencing and polymerase chain reaction assisted by gold nanoparticles, it is possible to identify several DNA variations that are associated with age-related macular degeneration and psoriasis on significant regions of human genomic DNA. Our method is accurate and promising for large-scale and high-throughput genetic analysis of susceptibility towards disease and drug resistance.

Vitamins E and C Prevent DNA Double-strand Breaks in Peripheral Lymphocytes Exposed to Radiations from Iodine-131.

PubMed

Safaei, Mehdi; Jafarpour, Seyed Masoud; Mohseni, Mehran; Salimian, Morteza; Akbari, Hossein; Karami, Fateme; Aliasgharzadeh, Akbar; Farhood, Bagher

2018-01-01

Iodine-131 is used as a radiopharmaceutical to treat thyroid cancer. The current study aimed to evaluate the effects of vitamins E and C on the level of DNA double-strand breaks (DSBs) caused by Radioiodine-131 (I-131) in human lymphocytes. Whole blood samples from human volunteers were incubated with a certain concentration of vitamins. After 1-h incubation, the samples were incubated with 20 μCi I-131/2 mL (blood + NaCl) for 1 h. To evaluate the effects of antioxidants, lymphocytes were separated, and the mean DSBs/cell was measured for each sample through γ-H2AX assay. After 1-h incubation with 20 μCi I-131/2 mL (blood + NaCl), iodine-131 increased the level of DSBs by 102.9%, compared with the background group. Vitamins E and C reduced the level of DSBs by 21.5% and 36.4%, respectively. Using vitamins E and C as antioxidants can reduce the toxicity of I-131. Furthermore, vitamin C provided the more protection for DNA, compared with vitamin E.

A low density microarray method for the identification of human papillomavirus type 18 variants.

PubMed

Meza-Menchaca, Thuluz; Williams, John; Rodríguez-Estrada, Rocío B; García-Bravo, Aracely; Ramos-Ligonio, Ángel; López-Monteon, Aracely; Zepeda, Rossana C

2013-09-26

We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18's variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings.

A Low Density Microarray Method for the Identification of Human Papillomavirus Type 18 Variants

PubMed Central

Meza-Menchaca, Thuluz; Williams, John; Rodríguez-Estrada, Rocío B.; García-Bravo, Aracely; Ramos-Ligonio, Ángel; López-Monteon, Aracely; Zepeda, Rossana C.

2013-01-01

We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18's variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings. PMID:24077317

PCR tools for the verification of the specific identity of ascaridoid nematodes from dogs and cats.

PubMed

Li, M W; Lin, R Q; Chen, H H; Sani, R A; Song, H Q; Zhu, X Q

2007-01-01

Based on the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of Toxocara canis, Toxocara cati, Toxocara malaysiensis and Toxascaris leonina, specific forward primers were designed in the ITS-1 or ITS-2 for each of the four ascaridoid species of dogs and cats. These primers were used individually together with a conserved primer in the large subunit of rDNA to amplify partial ITS-1 and/or ITS-2 of rDNA from 107 DNA samples from ascaridoids from dogs and cats in China, Australia, Malaysia, England and the Netherlands. This approach allowed their specific identification, with no amplicons being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the sequences amplified. The minimum amounts of DNA detectable using the PCR assays were 0.13-0.54ng. These PCR assays should provide useful tools for the diagnosis and molecular epidemiological investigations of toxocariasis in humans and animals.

DNA data in criminal procedure in the European fundamental rights context.

PubMed

Soleto, Helena

2014-01-01

Despite being one of the most useful and reliable identification tools, DNA profiling in criminal procedure balances on the border between the limitation and violation of Fundamental Rights that can occur beginning with the collection of the sample, its analysis, and its use; and ending with its processing. Throughout this complex process, violation of human or fundamental rights -such as the right to physical and moral integrity, the right not to be subject to degrading treatment, the right not to incriminate oneself, the right to family privacy together with that of not incriminating descendants or relatives in general, the right to personal development and the right to informative self-determination- is possible. This article presents an analysis of all the above-mentioned DNA treating phases in criminal process in the light of possible violations of some Fundamental Rights, while at the same time discarding some of them on the basis of European human rights protection standards. As the case-law of the European Court of Human Rights shows, the legislation on DNA collection and DNA related data processing or its implementation does not always respect all human rights and should be carefully considered before its adoption and during its application.

High Prevalence of Intermediate Leptospira spp. DNA in Febrile Humans from Urban and Rural Ecuador.

PubMed

Chiriboga, Jorge; Barragan, Verónica; Arroyo, Gabriela; Sosa, Andrea; Birdsell, Dawn N; España, Karool; Mora, Ana; Espín, Emilia; Mejía, María Eugenia; Morales, Melba; Pinargote, Carmina; Gonzalez, Manuel; Hartskeerl, Rudy; Keim, Paul; Bretas, Gustavo; Eisenberg, Joseph N S; Trueba, Gabriel

2015-12-01

Leptospira spp., which comprise 3 clusters (pathogenic, saprophytic, and intermediate) that vary in pathogenicity, infect >1 million persons worldwide each year. The disease burden of the intermediate leptospires is unclear. To increase knowledge of this cluster, we used new molecular approaches to characterize Leptospira spp. in 464 samples from febrile patients in rural, semiurban, and urban communities in Ecuador; in 20 samples from nonfebrile persons in the rural community; and in 206 samples from animals in the semiurban community. We observed a higher percentage of leptospiral DNA-positive samples from febrile persons in rural (64%) versus urban (21%) and semiurban (25%) communities; no leptospires were detected in nonfebrile persons. The percentage of intermediate cluster strains in humans (96%) was higher than that of pathogenic cluster strains (4%); strains in animal samples belonged to intermediate (49%) and pathogenic (51%) clusters. Intermediate cluster strains may be causing a substantial amount of fever in coastal Ecuador.

Use of DNA from human stools to detect aberrant CpG island methylation of genes implicated in colorectal cancer.

PubMed

Belshaw, Nigel J; Elliott, Giles O; Williams, Elizabeth A; Bradburn, David M; Mills, Sarah J; Mathers, John C; Johnson, Ian T

2004-09-01

Hypermethylation of cytosine residues in the CpG islands of tumor suppressor genes is a key mechanism of colorectal carcinogenesis. Detection and quantification of CpG island methylation in human DNA isolated from stools might provide a novel strategy for the detection and investigation of colorectal neoplasia. To explore the feasibility of this approach, colorectal biopsies and fecal samples were obtained from 32 patients attending for colonoscopy or surgery, who were found to have adenomatous polyps, colorectal cancer, or no evidence of neoplasia. A further 18 fecal samples were obtained from healthy volunteers, with no bowel symptoms. Isolated DNA was modified with sodium bisulfite and analyzed by methylation-specific PCR and combined bisulfite restriction analysis for CpG island methylation of ESR1, MGMT, HPP1, p16(INK4a), APC, and MLH1. CpG island methylation was readily detectable in both mucosal and fecal DNA with methylation-specific PCR. Using combined bisulfite restriction analysis, it was established that, in volunteers from whom biopsies were available, the levels of methylation at two CpG sites within ESR1 assayed using fecal DNA were significantly correlated with methylation in DNA from colorectal mucosa. Thus, noninvasive techniques can be used to obtain quantitative information about the level of CpG island methylation in human colorectal mucosa. The methods described here could be applied to a much expanded range of genes and may be valuable both for screening purposes and to provide greater insight into the functional consequences of epigenetic changes in the colorectal mucosa of free-living individuals.

Real-Time PCR with an Internal Control for Detection of All Known Human Adenovirus Serotypes▿

PubMed Central

Damen, Marjolein; Minnaar, René; Glasius, Patricia; van der Ham, Alwin; Koen, Gerrit; Wertheim, Pauline; Beld, Marcel

2008-01-01

The “gold standard” for the diagnosis of adenovirus (AV) infection is virus culture, which is rather time-consuming. Especially for immunocompromised patients, in whom severe infections with AV have been described, rapid diagnosis is important. Therefore, an internally controlled AV real-time PCR assay detecting all known human AV serotypes was developed. Primers were chosen from the hexon region, which is the most conserved region, and in order to cover all known serotypes, degenerate primers were used. The internal control (IC) DNA contained the same primer binding sites as the AV DNA control but had a shuffled probe region compared to the conserved 24-nucleotide consensus AV hexon probe region (the target). The IC DNA was added to the clinical sample in order to monitor extraction and PCR efficiency. The sensitivity and the linearity of the AV PCR were determined. For testing the specificity of this PCR assay for human AVs, a selection of 51 AV prototype strains and 66 patient samples positive for other DNA viruses were tested. Moreover, a comparison of the AV PCR method described herein with culture and antigen (Ag) detection was performed with a selection of 151 clinical samples. All 51 AV serotypes were detected in the selection of AV prototype strains. Concordant results from culture or Ag detection and PCR were found for 139 (92.1%) of 151 samples. In 12 cases (7.9%), PCR was positive while the culture was negative. In conclusion, a sensitive, internally controlled nonnested AV real-time PCR assay which is able to detect all known AV serotypes with higher sensitivity than a culture or Ag detection method was developed. PMID:18923006

THE USE OF MULTIPLE DISPLACEMENT AMPLIFICATION TO INCREASE THE DETECTION AND GENOTYPING OF TRYPANOSOMA SPECIES SAMPLES IMMOBILISED ON FTA FILTERS

PubMed Central

MORRISON, LIAM J.; McCORMACK, GILLIAN; SWEENEY, LINDSAY; LIKEUFACK, ANNE C. L.; TRUC, PHILIPPE; TURNER, C. MICHAEL; TAIT, ANDY; MacLEOD, ANNETTE

2007-01-01

Whole genome amplification methods are a recently developed tool for amplifying DNA from limited template. We report its application in trypanosome infections, characterised by low parasitaemias. Multiple Displacement Amplification (MDA) amplifies DNA with a simple in vitro step, and was evaluated on mouse blood samples on FTA filter cards with known numbers of Trypanosoma brucei parasites. The data showed a twenty-fold increase in the number of PCRs possible per sample, using primers diagnostic for the multi-copy ribosomal ITS region or 177 bp repeats, and a twenty-fold increase in sensitivity over nested PCR against a single copy microsatellite. Using MDA for microsatellite genotyping caused allele dropout at low DNA concentrations, which was overcome by pooling multiple MDA reactions. The validity of using MDA was established with samples from Human African Trypanosomiasis patients. The use of MDA allows maximal use of finite DNA samples and may prove a valuable tool in studies where multiple reactions are necessary, such as population genetic analyses. PMID:17556624

Direct and quantitative detection of HIV-1 RNA in human plasma with a branched DNA signal amplification assay.

PubMed

Urdea, M S; Wilber, J C; Yeghiazarian, T; Todd, J A; Kern, D G; Fong, S J; Besemer, D; Hoo, B; Sheridan, P J; Kokka, R

1993-11-01

To determine the relative effect of sample matrix on the quantitation of HIV RNA in plasma. Two HIV-positive specimens were diluted into five and 10 different HIV-negative plasma samples, respectively. Branched DNA signal amplification technology and reverse-transcriptase polymerase chain reaction were used to measure the viral load. In one sample the viral load by polymerase chain reaction ranged from undetectable to 1.9 x 10(5) copies/ml, and the branched DNA results ranged from 2.6 x 10(4) to 4.2 x 10(4) HIV RNA equivalent/ml. In the other sample the corresponding figures were 6.3 x 10(4) to 5.5 x 10(5) copies/ml and 5.7 x 10(4) to 7.5 x 10(4) HIV RNA equivalents/ml. In contrast to reverse-transcriptase polymerase chain reaction the branched DNA signal amplification assay does not require a separate extraction step or enzymatic amplification of the target. Therefore this measurement is less affected by the sample matrix and the signal generated is directly proportional to the viral load.

Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

NASA Astrophysics Data System (ADS)

Ma, Yunfei; Zhang, Honglian; Liu, Fangming; Wu, Zhenhua; Lu, Shaohua; Jin, Qinghui; Zhao, Jianlong; Zhong, Xinhua; Mao, Hongju

2015-10-01

DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers.DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers. Electronic supplementary information (ESI) available: Synthesis of CdSe/CdS/ZnS core/shell/shell QDs. Sequences of primers used for amplifying the promoter regions in bisulfate-modified DNA. Comparison of detected methylation levels in different gene promoters using the QD-based FRET method versus bisulfite pyrosequencing. Methylation levels of the RASSF1A gene in one pair of NT and cancer samples as indicated by pyrosequencing. Theoretical calculation of the Förster distance R0. See DOI: 10.1039/c5nr04956c

Assay for identification of heterozygous single-nucleotide polymorphism (Ala67Thr) in human poliovirus receptor gene.

PubMed

Nandi, Shyam Sundar; Sharma, Deepa Kailash; Deshpande, Jagadish M

2016-07-01

It is important to understand the role of cell surface receptors in susceptibility to infectious diseases. CD155 a member of the immunoglobulin super family, serves as the poliovirus receptor (PVR). Heterozygous (Ala67Thr) polymorphism in CD155 has been suggested as a risk factor for paralytic outcome of poliovirus infection. The present study pertains to the development of a screening test to detect the single nucleotide (SNP) polymorphism in the CD155 gene. New primers were designed for PCR, sequencing and SNP analysis of Exon2 of CD155 gene. DNAs extracted from either whole blood (n=75) or cells from oral cavity (n=75) were used for standardization and validation of the SNP assay. DNA sequencing was used as the gold standard method. A new SNP assay for detection of heterozygous Ala67Thr genotype was developed and validated by testing 150 DNA samples. Heterozygous CD155 was detected in 27.33 per cent (41/150) of DNA samples tested by both SNP detection assay and sequencing. The SNP detection assay was successfully developed for identification of Ala67Thr polymorphism in human PVR/CD155 gene. The SNP assay will be useful for large scale screening of DNA samples.

Identification of tissue-specific cell death using methylation patterns of circulating DNA

PubMed Central

Lehmann-Werman, Roni; Neiman, Daniel; Zemmour, Hai; Moss, Joshua; Magenheim, Judith; Vaknin-Dembinsky, Adi; Rubertsson, Sten; Nellgård, Bengt; Blennow, Kaj; Zetterberg, Henrik; Spalding, Kirsty; Haller, Michael J.; Wasserfall, Clive H.; Schatz, Desmond A.; Greenbaum, Carla J.; Dorrell, Craig; Grompe, Markus; Zick, Aviad; Hubert, Ayala; Maoz, Myriam; Fendrich, Volker; Bartsch, Detlef K.; Golan, Talia; Ben Sasson, Shmuel A.; Zamir, Gideon; Razin, Aharon; Cedar, Howard; Shapiro, A. M. James; Glaser, Benjamin; Shemer, Ruth; Dor, Yuval

2016-01-01

Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics. PMID:26976580

Multiplex quantification of 16S rDNA of predominant bacteria group within human fecal samples by polymerase chain reaction--ligase detection reaction (PCR-LDR).

PubMed

Li, Kai; Chen, Bei; Zhou, Yuxun; Huang, Rui; Liang, Yinming; Wang, Qinxi; Xiao, Zhenxian; Xiao, Junhua

2009-03-01

A new method, based on ligase detection reaction (LDR), was developed for quantitative detection of multiplex PCR amplicons of 16S rRNA genes present in complex mixtures (specifically feces). LDR has been widely used in single nucleotide polymorphism (SNP) assay but never applied for quantification of multiplex PCR products. This method employs one pair of DNA probes, one of which is labeled with fluorescence for signal capture, complementary to the target sequence. For multiple target sequence analysis, probes were modified with different lengths of polyT at the 5' end and 3' end. Using a DNA sequencer, these ligated probes were separated and identified by size and dye color. Then, relative abundance of target DNA were normalized and quantified based on the fluorescence intensities and exterior size standards. 16S rRNA gene of three preponderant bacteria groups in human feces: Clostridium coccoides, Bacteroides and related genera, and Clostridium leptum group, were amplified and cloned into plasmid DNA so as to make standard curves. After PCR-LDR analysis, a strong linear relationship was found between the florescence intensity and the diluted plasmid DNA concentrations. Furthermore, based on this method, 100 human fecal samples were quantified for the relative abundance of the three bacterial groups. Relative abundance of C. coccoides was significantly higher in elderly people in comparison with young adults, without gender differences. Relative abundance of Bacteroides and related genera and C. leptum group were significantly higher in young and middle aged than in the elderly. Regarding the whole set of sample, C. coccoides showed the highest relative abundance, followed by decreasing groups Bacteroides and related genera, and C. leptum. These results imply that PCR-LDR can be feasible and flexible applied to large scale epidemiological studies.

The common occurrence of epistasis in the determination of human pigmentation and its impact on DNA-based pigmentation phenotype prediction.

PubMed

Pośpiech, Ewelina; Wojas-Pelc, Anna; Walsh, Susan; Liu, Fan; Maeda, Hitoshi; Ishikawa, Takaki; Skowron, Małgorzata; Kayser, Manfred; Branicki, Wojciech

2014-07-01

The role of epistatic effects in the determination of complex traits is often underlined but its significance in the prediction of pigmentation phenotypes has not been evaluated so far. The prediction of pigmentation from genetic data can be useful in forensic science to describe the physical appearance of an unknown offender, victim, or missing person who cannot be identified via conventional DNA profiling. Available forensic DNA prediction systems enable the reliable prediction of several eye and hair colour categories. However, there is still space for improvement. Here we verified the association of 38 candidate DNA polymorphisms from 13 genes and explored the extent to which interactions between them may be involved in human pigmentation and their impact on forensic DNA prediction in particular. The model-building set included 718 Polish samples and the model-verification set included 307 independent Polish samples and additional 72 samples from Japan. In total, 29 significant SNP-SNP interactions were found with 5 of them showing an effect on phenotype prediction. For predicting green eye colour, interactions between HERC2 rs12913832 and OCA2 rs1800407 as well as TYRP1 rs1408799 raised the prediction accuracy expressed by AUC from 0.667 to 0.697 and increased the prediction sensitivity by >3%. Interaction between MC1R 'R' variants and VDR rs731236 increased the sensitivity for light skin by >1% and by almost 3% for dark skin colour prediction. Interactions between VDR rs1544410 and TYR rs1042602 as well as between MC1R 'R' variants and HERC2 rs12913832 provided an increase in red/non-red hair prediction accuracy from an AUC of 0.902-0.930. Our results thus underline epistasis as a common phenomenon in human pigmentation genetics and demonstrate that considering SNP-SNP interactions in forensic DNA phenotyping has little impact on eye, hair and skin colour prediction. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Improved methods of DNA extraction from human spermatozoa that mitigate experimentally-induced oxidative DNA damage.

PubMed

Xavier, Miguel J; Nixon, Brett; Roman, Shaun D; Aitken, Robert John

2018-01-01

Current approaches for DNA extraction and fragmentation from mammalian spermatozoa provide several challenges for the investigation of the oxidative stress burden carried in the genome of male gametes. Indeed, the potential introduction of oxidative DNA damage induced by reactive oxygen species, reducing agents (dithiothreitol or beta-mercaptoethanol), and DNA shearing techniques used in the preparation of samples for chromatin immunoprecipitation and next-generation sequencing serve to cofound the reliability and accuracy of the results obtained. Here we report optimised methodology that minimises, or completely eliminates, exposure to DNA damaging compounds during extraction and fragmentation procedures. Specifically, we show that Micrococcal nuclease (MNase) digestion prior to cellular lysis generates a greater DNA yield with minimal collateral oxidation while randomly fragmenting the entire paternal genome. This modified methodology represents a significant improvement over traditional fragmentation achieved via sonication in the preparation of genomic DNA from human spermatozoa for downstream applications, such as next-generation sequencing. We also present a redesigned bioinformatic pipeline framework adjusted to correctly analyse this form of data and detect statistically relevant targets of oxidation.

Searching beyond the usual papillomavirus suspects in squamous carcinomas of the vulva, penis and head and neck.

PubMed

Félez-Sánchez, Marta; Vergara, Marleny; de Sanjosé, Silvia; Castellsagué, Xavier; Alemany, Laia; Bravo, Ignacio G

2016-11-01

Human Papillomaviruses (HPVs) are involved in the etiology of anogenital and head and neck cancers. The HPV DNA prevalence greatly differs by anatomical site. Indeed, the high rates of viral DNA prevalence in anal and cervical carcinomas contrast with the lower fraction of cancer cases attributable to HPVs in other anatomical sites, chiefly the vulva, the penis and head and neck. Here we analyzed 2635 Formalin Fixed Paraffin Embedded surgical samples that had previously tested negative for the presence of HPVs DNA using the SPF10/DEIA procedure, in order to identify the presence of other PVs not explicitly targeted by standard molecular epidemiologic approaches. All samples were reanalyzed using five broad-PV PCR primer sets (CP1/2, FAP6064/FAP64, SKF/SKR, MY9/MY11, MFI/MFII) targeting the main PV main clades. In head and neck carcinoma samples (n=1141), we recovered DNA from two BetaHPVs, namely HPV20 and HPV21, and from three cutaneous AlphaPVs, namely HPV2, HPV57 and HPV61. In vulvar squamous cell carcinoma samples (n=902), we found one of the samples containing DNA of one cutaneous HPV, namely HPV2, and 29 samples contained DNA from essentially mucosal HPVs. In penile squamous cell carcinoma samples (n=592), we retrieved the DNA of HPV16 in 16 samples. Our results show first that the SPF10/DEIA is very sensitive, as we recovered only 2.1% (55/2635) false negative results; second, that although the DNA of cutaneous HPVs can be detected in cancer samples, their relative contribution remains anyway minor (0.23%; 6/2635) and may be neglected for screening and vaccination purposes; and third, their contribution to malignancy is not necessarily warranted and needs to be elucidated. Copyright © 2016 Elsevier B.V. All rights reserved.

Differential Nuclear and Mitochondrial DNA Preservation in Post-Mortem Teeth with Implications for Forensic and Ancient DNA Studies

PubMed Central

Higgins, Denice; Rohrlach, Adam B.; Kaidonis, John; Townsend, Grant; Austin, Jeremy J.

2015-01-01

Major advances in genetic analysis of skeletal remains have been made over the last decade, primarily due to improvements in post-DNA-extraction techniques. Despite this, a key challenge for DNA analysis of skeletal remains is the limited yield of DNA recovered from these poorly preserved samples. Enhanced DNA recovery by improved sampling and extraction techniques would allow further advancements. However, little is known about the post-mortem kinetics of DNA degradation and whether the rate of degradation varies between nuclear and mitochondrial DNA or across different skeletal tissues. This knowledge, along with information regarding ante-mortem DNA distribution within skeletal elements, would inform sampling protocols facilitating development of improved extraction processes. Here we present a combined genetic and histological examination of DNA content and rates of DNA degradation in the different tooth tissues of 150 human molars over short-medium post-mortem intervals. DNA was extracted from coronal dentine, root dentine, cementum and pulp of 114 teeth via a silica column method and the remaining 36 teeth were examined histologically. Real time quantification assays based on two nuclear DNA fragments (67 bp and 156 bp) and one mitochondrial DNA fragment (77 bp) showed nuclear and mitochondrial DNA degraded exponentially, but at different rates, depending on post-mortem interval and soil temperature. In contrast to previous studies, we identified differential survival of nuclear and mtDNA in different tooth tissues. Futhermore histological examination showed pulp and dentine were rapidly affected by loss of structural integrity, and pulp was completely destroyed in a relatively short time period. Conversely, cementum showed little structural change over the same time period. Finally, we confirm that targeted sampling of cementum from teeth buried for up to 16 months can provide a reliable source of nuclear DNA for STR-based genotyping using standard extraction methods, without the need for specialised equipment or large-volume demineralisation steps. PMID:25992635

Evaluating the Impact of DNA Extraction Method on the Representation of Human Oral Bacterial and Fungal Communities

PubMed Central

Biswas, Kristi; Taylor, Michael W.; Gear, Kim

2017-01-01

The application of high-throughput, next-generation sequencing technologies has greatly improved our understanding of the human oral microbiome. While deciphering this diverse microbial community using such approaches is more accurate than traditional culture-based methods, experimental bias introduced during critical steps such as DNA extraction may compromise the results obtained. Here, we systematically evaluate four commonly used microbial DNA extraction methods (MoBio PowerSoil® DNA Isolation Kit, QIAamp® DNA Mini Kit, Zymo Bacterial/Fungal DNA Mini PrepTM, phenol:chloroform-based DNA isolation) based on the following criteria: DNA quality and yield, and microbial community structure based on Illumina amplicon sequencing of the V3–V4 region of the 16S rRNA gene of bacteria and the internal transcribed spacer (ITS) 1 region of fungi. Our results indicate that DNA quality and yield varied significantly with DNA extraction method. Representation of bacterial genera in plaque and saliva samples did not significantly differ across DNA extraction methods and DNA extraction method showed no effect on the recovery of fungal genera from plaque. By contrast, fungal diversity from saliva was affected by DNA extraction method, suggesting that not all protocols are suitable to study the salivary mycobiome. PMID:28099455

Forensic DNA Phenotyping: Predicting human appearance from crime scene material for investigative purposes.

PubMed

Kayser, Manfred

2015-09-01

Forensic DNA Phenotyping refers to the prediction of appearance traits of unknown sample donors, or unknown deceased (missing) persons, directly from biological materials found at the scene. "Biological witness" outcomes of Forensic DNA Phenotyping can provide investigative leads to trace unknown persons, who are unidentifiable with current comparative DNA profiling. This intelligence application of DNA marks a substantially different forensic use of genetic material rather than that of current DNA profiling presented in the courtroom. Currently, group-specific pigmentation traits are already predictable from DNA with reasonably high accuracies, while several other externally visible characteristics are under genetic investigation. Until individual-specific appearance becomes accurately predictable from DNA, conventional DNA profiling needs to be performed subsequent to appearance DNA prediction. Notably, and where Forensic DNA Phenotyping shows great promise, this is on a (much) smaller group of potential suspects, who match the appearance characteristics DNA-predicted from the crime scene stain or from the deceased person's remains. Provided sufficient funding being made available, future research to better understand the genetic basis of human appearance will expectedly lead to a substantially more detailed description of an unknown person's appearance from DNA, delivering increased value for police investigations in criminal and missing person cases involving unknowns. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Evaluation of Buccal Cell Samples for Studies of Oral Microbiota.

PubMed

Yu, Guoqin; Phillips, Steve; Gail, Mitchell H; Goedert, James J; Humphrys, Michael; Ravel, Jacques; Ren, Yanfang; Caporaso, Neil E

2017-02-01

The human microbiota is postulated to affect cancer risk, but collecting microbiota specimens with prospective follow-up for diseases will take time. Buccal cell samples have been obtained from mouthwash for the study of human genomic DNA in many cohort studies. Here, we evaluate the feasibility of using buccal cell samples to examine associations of human microbiota and disease risk. We obtained buccal cells from mouthwash in 41 healthy participants using a protocol that is widely employed to obtain buccal cells for the study of human DNA. We compared oral microbiota from buccal cells with that from eight other oral sample types collected by following the protocols of the Human Microbiome Project. Microbiota profiles were determined by sequencing 16S rRNA gene V3-V4 region. Compared with each of the eight other oral samples, the buccal cell samples had significantly more observed species (P < 0.002) and higher alpha diversity (Shannon index, P < 0.02). The microbial communities were more similar (smaller beta diversity) among buccal cells samples than in the other samples (P < 0.001 for 12 of 16 weighted and unweighted UniFrac distance comparisons). Buccal cell microbial profiles closely resembled saliva but were distinct from dental plaque and tongue dorsum. Stored buccal cell samples in prospective cohort studies are a promising resource to study associations of oral microbiota with disease. The feasibility of using existing buccal cell collections in large prospective cohorts allows investigations of the role of oral microbiota in chronic disease etiology in large population studies possible today. Cancer Epidemiol Biomarkers Prev; 26(2); 249-53. ©2016 AACR. ©2016 American Association for Cancer Research.

Direct PCR amplification of DNA from human bloodstains, saliva, and touch samples collected with microFLOQ® swabs.

PubMed

Ambers, Angie; Wiley, Rachel; Novroski, Nicole; Budowle, Bruce

2018-01-01

Previous studies have shown that nylon flocked swabs outperform traditional fiber swabs in DNA recovery due to their innovative design and lack of internal absorbent core to entrap cellular materials. The microFLOQ ® Direct swab, a miniaturized version of the 4N6 FLOQSwab ® , has a small swab head that is treated with a lysing agent which allows for direct amplification and DNA profiling from sample collection to final result in less than two hours. Additionally, the microFLOQ ® system subsamples only a minute portion of a stain and preserves the vast majority of the sample for subsequent testing or re-analysis, if desired. The efficacy of direct amplification of DNA from dilute bloodstains, saliva stains, and touch samples was evaluated using microFLOQ ® Direct swabs and the GlobalFiler™ Express system. Comparisons were made to traditional methods to assess the robustness of this alternate workflow. Controlled studies with 1:19 and 1:99 dilutions of bloodstains and saliva stains consistently yielded higher STR peak heights than standard methods with 1ng input DNA from the same samples. Touch samples from common items yielded single source and mixed profiles that were consistent with primary users of the objects. With this novel methodology/workflow, no sample loss occurs and therefore more template DNA is available during amplification. This approach may have important implications for analysis of low quantity and/or degraded samples that plague forensic casework. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Detection of human papillomavirus and Epstein-Barr virus DNA sequences in oral mucosa of HIV-infected patients by the polymerase chain reaction.

PubMed Central

Snijders, P. J.; Schulten, E. A.; Mullink, H.; ten Kate, R. W.; Jiwa, M.; van der Waal, I.; Meijer, C. J.; Walboomers, J. M.

1990-01-01

The presence of human papillomavirus (HPV) and Epstein-Barr virus (EBV) was analyzed in 21 oral biopsy specimens of HIV-infected patients using the polymerase chain reaction (PCR) method. Biopsies were categorized as hairy leukoplakia (HL) (n = 12), candidiasis (n = 3), oral warts (n = 2), and clinically normal epithelium (n = 4). For HPV detection a modified general primer-mediated PCR method (GP-PCR), which detects a broad spectrum of HPV genotypes at sub-picogram levels, was used. Human papillomavirus DNA was only found in two oral warts and was identified as HPV type 32. Epstein-Barr virus DNA was detected in 16 biopsy specimens, including the 12 HLs, 2 cases of candidiasis, and 2 samples of normal epithelium. Epstein-Barr virus positivity in HL could be confirmed by Southern blot analysis and DNA in situ hybridization using biotinylated DNA probes (bio-DISH). Epstein-Barr virus bio-DISH was also positive in one sample of normal epithelium from a patient with HL. The results indicate that HL is strongly associated with EBV and not with any of the common HPV types that react with general HPV primers in the PCR. However the detection of EBV in normal oral epithelium by PCR and bio-DISH suggests that the presence of this virus is not exclusively related to HL. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:2169191

Forensic genetic analysis of bone remain samples.

PubMed

Siriboonpiputtana, T; Rinthachai, T; Shotivaranon, J; Peonim, V; Rerkamnuaychoke, B

2018-03-01

DNA typing from degraded human remains is still challenging forensic DNA scientists not only in the prospective of DNA purification but also in the interpretation of established DNA profiles and data manipulation, especially in mass fatalities. In this report, we presented DNA typing protocol to investigate many skeletal remains in different degrees of decomposing. In addition, we established the grading system aiming for prior determination of the association between levels of decomposing and overall STR amplification efficacy. A total of 80 bone samples were subjected to DNA isolation using the modified DNA IQ™ System (Promega, USA) for bone extraction following with STR analysis using the AmpFLSTR Identifiler ® (Thermo Fisher Scientific, USA). In low destruction group, complete STR profiles were observed as 84.4% whereas partial profiles and non-amplified were found as 9.4% and 6.2%, respectively. Moreover, in medium destruction group, both complete and partial STR profiles were observed as 31.2% while 37.5% of this group was unable to amplify. Nevertheless, we could not purify DNA and were unable to generate STR profile in any sample from the high destroyed bone samples. Compact bones such as femur and humerus have high successful amplification rate superior than loose/spongy bones. Furthermore, costal cartilage could be a designate specimen for DNA isolation in a case of the body that was discovered approximately to 3 days after death which enabled to isolate high quality and quantity of DNA, reduce time and cost, and do not require special tools such as freezer mill. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of Skeletal Remains of Communist Armed Forces Victims During and After World War II: Combined Y-chromosome Short Tandem Repeat (STR) and MiniSTR Approach

PubMed Central

Marjanović, Damir; Durmić-Pašić, Adaleta; Kovačević, Lejla; Avdić, Jasna; Džehverović, Mirela; Haverić, Sanin; Ramić, Jasmin; Kalamujić, Belma; Bilela, Lada Lukić; Škaro, Vedrana; Projić, Petar; Bajrović, Kasim; Drobnič, Katja; Davoren, Jon; Primorac, Dragan

2009-01-01

Aim To report on the use of STR, Y-STRs, and miniSTRs typing methods in the identification of victims of revolutionary violence and crimes against humanity committed by the Communist Armed Forces during and after World War II in which bodies were exhumed from mass and individual graves in Slovenia. Methods Bone fragments and teeth were removed from human remains found in several small and closely located hidden mass graves in the Škofja Loka area (Lovrenska Grapa and Žolšče) and 2 individual graves in the Ljubljana area (Podlipoglav), Slovenia. DNA was isolated using the Qiagen DNA extraction procedure optimized for bone and teeth. Some DNA extracts required additional purification, such as N-buthanol treatment. The QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. Initially, PowerPlex 16 kit was used to simultaneously analyze 15 short tandem repeat (STR) loci. The PowerPlex S5 miniSTR kit and AmpFℓSTR® MiniFiler PCR Amplification Kit was used for additional analysis if preliminary analysis yielded weak partial or no profiles at all. In 2 cases, when the PowerPlex 16 profiles indicated possible relatedness of the remains with reference samples, but there were insufficient probabilities to call the match to possible male paternal relatives, we resorted to an additional analysis of Y-STR markers. PowerPlex® Y System was used to simultaneously amplify 12 Y-STR loci. Fragment analysis was performed on an ABI PRISM 310 genetic analyzer. Matching probabilities were estimated using the DNA-View software. Results Following the Y-STR analysis, 1 of the “weak matches” previously obtained based on autosomal loci, was confirmed while the other 1 was not. Combined standard STR and miniSTR approach applied to bone samples from 2 individual graves resulted in positive identifications. Finally, using the same approach on 11 bone samples from hidden mass grave Žološče, we were able to obtain 6 useful DNA profiles. Conclusion The results of this study, in combination with previously obtained results, demonstrate that Y-chromosome testing and miniSTR methodology can contribute to the identification of human remains of victims of revolutionary violence from World War II. PMID:19480024

[Study on the infection of taiga ticks with Borrelia in the territory of Novosibirsk Scientific Center SB PAS].

PubMed

Borgoiakov, V Iu; Fomenko, N V; Panov, V V; Chikova, E D

2010-01-01

In our study, Borrelia were revealed in the taiga ticks Ixodes persulcatus collected on vegetation by flagging, as well as in the ticks removed from the people who asked for help in the vaccination center located in the Novosibirsk Scientific Center of the Siberian Branch of Russian Academy of Science (NS SB RAS). By the isolation of Borrelia on BSK-H medum, the occurrence of B. garinii, B. afzelii, and B. miyamotoi was established in the territory of NSC. B. miyamotoi isolates were unstable and lost their ability to growth in later passages. DNA of the same three species of Borrelia was detected by PCR in the samples of ticks, both collected on vegetation by flagging and removed from humans. DNA of B. garinii was recorded most often; DNA of B. afzelii was less frequent; and the least number of positive samples was shown for B. miyamotoi. In the ticks collected on vegetation by flagging, DNA of B. garinii was found in 38.6%, B. afzelii in 9.9%, and B. miyamoboi in 3.9% of samples. In the ticks removed from people, number of positive samples was lesser; so, DNA of B. garinii was detected in 24.2%, B. afzelii in 6.9%, and B. miyamotoi in 5.6% of samples. Mixed infection with two Borrelia species was recorded, and DNA of B. mivamnotoi more often detected simultaneously with DNA of B. garinii.

Constructing DNA Barcode Sets Based on Particle Swarm Optimization.

PubMed

Wang, Bin; Zheng, Xuedong; Zhou, Shihua; Zhou, Changjun; Wei, Xiaopeng; Zhang, Qiang; Wei, Ziqi

2018-01-01

Following the completion of the human genome project, a large amount of high-throughput bio-data was generated. To analyze these data, massively parallel sequencing, namely next-generation sequencing, was rapidly developed. DNA barcodes are used to identify the ownership between sequences and samples when they are attached at the beginning or end of sequencing reads. Constructing DNA barcode sets provides the candidate DNA barcodes for this application. To increase the accuracy of DNA barcode sets, a particle swarm optimization (PSO) algorithm has been modified and used to construct the DNA barcode sets in this paper. Compared with the extant results, some lower bounds of DNA barcode sets are improved. The results show that the proposed algorithm is effective in constructing DNA barcode sets.

From the mouths of monkeys: Detection of Mycobacterium tuberculosis complex DNA from buccal swabs of synanthropic macaques

PubMed Central

Wilbur, AK; Engel, G; Rompis, A; Putra, IGA A; Lee, BP Y-H; Aggimarangsee, N; Chalise, M; Shaw, E; Oh, G; Schillaci, MA; Jones-Engel, L

2012-01-01

Although the Mycobacterium tuberculosis complex (MTBC) infects a third of all humans, little is known regarding the prevalence of mycobacterial infection in nonhuman primates (NHP). For more than a century, tuberculosis has been regarded as a serious infectious threat to NHP species. Advances in the detection of MTBC open new possibilities for investigating the effects of this poorly understood pathogen in diverse populations of NHP. Here we report results of a cross-sectional study using well-described molecular methods to detect a nucleic acid sequence (IS6110) unique to the MTBC. Sample collection was focused on the oral cavity, the presumed route of transmission of MTBC. Buccal swabs were collected from 263 macaques representing 11 species in four Asian countries and Gibraltar. Contexts of contact with humans included free ranging, pets, performing monkeys, zoos, and monkey temples. Following DNA isolation from buccal swabs, the PCR amplified IS6110 from 84 (31.9%) of the macaques. In general, prevalence of MTBC DNA was higher among NHP in countries where the World Health Organization reports higher prevalence of humans infected with MTBC. This is the first demonstration of MTBC DNA in the mouths of macaques. Further research is needed to establish the significance of this finding at both the individual and population levels. PCR of buccal samples holds promise as a method to elucidate the mycobacterial landscape among NHP, particularly macaques that thrive in areas of high human MTBC prevalence. PMID:22644580

Use of laptop computers connected to internet through Wi-Fi decreases human sperm motility and increases sperm DNA fragmentation.

PubMed

Avendaño, Conrado; Mata, Ariela; Sanchez Sarmiento, César A; Doncel, Gustavo F

2012-01-01

To evaluate the effects of laptop computers connected to local area networks wirelessly (Wi-Fi) on human spermatozoa. Prospective in vitro study. Center for reproductive medicine. Semen samples from 29 healthy donors. Motile sperm were selected by swim up. Each sperm suspension was divided into two aliquots. One sperm aliquot (experimental) from each patient was exposed to an internet-connected laptop by Wi-Fi for 4 hours, whereas the second aliquot (unexposed) was used as control, incubated under identical conditions without being exposed to the laptop. Evaluation of sperm motility, viability, and DNA fragmentation. Donor sperm samples, mostly normozoospermic, exposed ex vivo during 4 hours to a wireless internet-connected laptop showed a significant decrease in progressive sperm motility and an increase in sperm DNA fragmentation. Levels of dead sperm showed no significant differences between the two groups. To our knowledge, this is the first study to evaluate the direct impact of laptop use on human spermatozoa. Ex vivo exposure of human spermatozoa to a wireless internet-connected laptop decreased motility and induced DNA fragmentation by a nonthermal effect. We speculate that keeping a laptop connected wirelessly to the internet on the lap near the testes may result in decreased male fertility. Further in vitro and in vivo studies are needed to prove this contention. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Effects of Anticoagulant, Processing Delay, and Assay Method (Branched DNA versus Reverse Transcriptase PCR) on Measurement of Human Immunodeficiency Virus Type 1 RNA Levels in Plasma

PubMed Central

Kirstein, Lynn M.; Mellors, John W.; Rinaldo, Charles R.; Margolick, Joseph B.; Giorgi, Janis V.; Phair, John P.; Dietz, Edith; Gupta, Phalguni; Sherlock, Christopher H.; Hogg, Robert; Montaner, J. S. G.; Muñoz, Alvaro

1999-01-01

We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (P < 0.05) but small after 6 h (bDNA assay, −0.12 log10 copies/ml; RT-PCR, −0.05 log10 copies/ml) and after 18 h (bDNA assay, −0.27 log10 copies/ml; RT-PCR, −0.15 log10 copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, −0.002 log10 copies/ml; RT-PCR, −0.02 log10 copies/ml), but it was after 18 h (bDNA assay, −0.09 log10 copies/ml; RT-PCR, −0.09 log10 copies/ml). Only 4% of samples processed after 6 h lost more than 50% (≥0.3 log10 copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4+-cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (−0.05 to −0.12 log10 copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4+-cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP. PMID:10405379

Encoding of low-quality DNA profiles as genotype probability matrices for improved profile comparisons, relatedness evaluation and database searches.

PubMed

Ryan, K; Williams, D Gareth; Balding, David J

2016-11-01

Many DNA profiles recovered from crime scene samples are of a quality that does not allow them to be searched against, nor entered into, databases. We propose a method for the comparison of profiles arising from two DNA samples, one or both of which can have multiple donors and be affected by low DNA template or degraded DNA. We compute likelihood ratios to evaluate the hypothesis that the two samples have a common DNA donor, and hypotheses specifying the relatedness of two donors. Our method uses a probability distribution for the genotype of the donor of interest in each sample. This distribution can be obtained from a statistical model, or we can exploit the ability of trained human experts to assess genotype probabilities, thus extracting much information that would be discarded by standard interpretation rules. Our method is compatible with established methods in simple settings, but is more widely applicable and can make better use of information than many current methods for the analysis of mixed-source, low-template DNA profiles. It can accommodate uncertainty arising from relatedness instead of or in addition to uncertainty arising from noisy genotyping. We describe a computer program GPMDNA, available under an open source licence, to calculate LRs using the method presented in this paper. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Protocol for the use of a rapid real-time PCR method for the detection of HIV-1 proviral DNA using double-stranded primer.

PubMed

Pau, Chou-Pong; Wells, Susan K; Granade, Timothy C

2012-01-01

This chapter describes a real-time PCR method for the detection of HIV-1 proviral DNA in whole blood samples using a novel double-stranded primer system. The assay utilizes a simple commercially available DNA extraction method and a rapid and easy-to-perform real-time PCR protocol to consistently detect a minimum of four copies of HIV-1 group M proviral DNA in as little as 90 min after sample (whole blood) collection. Co-amplification of the human RNase P gene serves as an internal control to monitor the efficiency of both the DNA extraction and amplification. Once the assay is validated properly, it may be suitable as an alternative confirmation test for HIV-1 infections in a variety of HIV testing venues including the mother-to-child transmission testing sites, clinics, and diagnostic testing centers.

Sequence analysis of the canine mitochondrial DNA control region from shed hair samples in criminal investigations.

PubMed

Berger, C; Berger, B; Parson, W

2012-01-01

In recent years, evidence from domestic dogs has increasingly been analyzed by forensic DNA testing. Especially, canine hairs have proved most suitable and practical due to the high rate of hair transfer occurring between dogs and humans. Starting with the description of a contamination-free sample handling procedure, we give a detailed workflow for sequencing hypervariable segments (HVS) of the mtDNA control region from canine evidence. After the hair material is lysed and the DNA extracted by Phenol/Chloroform, the amplification and sequencing strategy comprises the HVS I and II of the canine control region and is optimized for DNA of medium-to-low quality and quantity. The sequencing procedure is based on the Sanger Big-dye deoxy-terminator method and the separation of the sequencing reaction products is performed on a conventional multicolor fluorescence detection capillary electrophoresis platform. Finally, software-aided base calling and sequence interpretation are addressed exemplarily.

Usefulness of telomere length in DNA from human teeth for age estimation.

PubMed

Márquez-Ruiz, Ana Belén; González-Herrera, Lucas; Valenzuela, Aurora

2018-03-01

Age estimation is widely used to identify individuals in forensic medicine. However, the accuracy of the most commonly used procedures is markedly reduced in adulthood, and these methods cannot be applied in practice when morphological information is limited. Molecular methods for age estimation have been extensively developed in the last few years. The fact that telomeres shorten at each round of cell division has led to the hypothesis that telomere length can be used as a tool to predict age. The present study thus aimed to assess the correlation between telomere length measured in dental DNA and age, and the effect of sex and tooth type on telomere length; a further aim was to propose a statistical regression model to estimate the biological age based on telomere length. DNA was extracted from 91 tooth samples belonging to 77 individuals of both sexes and 15 to 85 years old and was used to determine telomere length by quantitative real-time PCR. Our results suggested that telomere length was not affected by sex and was greater in molar teeth. We found a significant correlation between age and telomere length measured in DNA from teeth. However, the equation proposed to predict age was not accurate enough for forensic age estimation on its own. Age estimation based on telomere length in DNA from tooth samples may be useful as a complementary method which provides an approximate estimate of age, especially when human skeletal remains are the only forensic sample available.

Microfluidic Devices for Forensic DNA Analysis: A Review.

PubMed

Bruijns, Brigitte; van Asten, Arian; Tiggelaar, Roald; Gardeniers, Han

2016-08-05

Microfluidic devices may offer various advantages for forensic DNA analysis, such as reduced risk of contamination, shorter analysis time and direct application at the crime scene. Microfluidic chip technology has already proven to be functional and effective within medical applications, such as for point-of-care use. In the forensic field, one may expect microfluidic technology to become particularly relevant for the analysis of biological traces containing human DNA. This would require a number of consecutive steps, including sample work up, DNA amplification and detection, as well as secure storage of the sample. This article provides an extensive overview of microfluidic devices for cell lysis, DNA extraction and purification, DNA amplification and detection and analysis techniques for DNA. Topics to be discussed are polymerase chain reaction (PCR) on-chip, digital PCR (dPCR), isothermal amplification on-chip, chip materials, integrated devices and commercially available techniques. A critical overview of the opportunities and challenges of the use of chips is discussed, and developments made in forensic DNA analysis over the past 10-20 years with microfluidic systems are described. Areas in which further research is needed are indicated in a future outlook.

Genotoxic effect of N-hydroxy-4-acetylaminobiphenyl on human DNA: implications in bladder cancer.

PubMed

Shahab, Uzma; Moinuddin; Ahmad, Saheem; Dixit, Kiran; Habib, Safia; Alam, Khursheed; Ali, Asif

2013-01-01

The interaction of environmental chemicals and their metabolites with biological macromolecules can result in cytotoxic and genotoxic effects. 4-Aminobiphenyl (4-ABP) and several other related arylamines have been shown to be causally involved in the induction of human urinary bladder cancers. The genotoxic and the carcinogenic effects of 4-ABP are exhibited only when it is metabolically converted to a reactive electrophile, the aryl nitrenium ions, which subsequently binds to DNA and induce lesions. Although several studies have reported the formation of 4-ABP-DNA adducts, no extensive work has been done to investigate the immunogenicity of 4-ABP-modified DNA and its possible involvement in the generation of antibodies in bladder cancer patients. Human DNA was modified by N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), a reactive metabolite of 4-ABP. Structural perturbations in the N-OH-AABP modified DNA were assessed by ultraviolet, fluorescence, and circular dichroic spectroscopy as well as by agarose gel electrophoresis. Genotoxicity of N-OH-AABP modified DNA was ascertained by comet assay. High performance liquid chromatography (HPLC) analysis of native and modified DNA samples confirmed the formation of N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-4ABP) in the N-OH-AABP damaged DNA. The experimentally induced antibodies against N-OH-AABP-modified DNA exhibited much better recognition of the DNA isolated from bladder cancer patients as compared to the DNA obtained from healthy individuals in competitive binding ELISA. This work shows epitope sharing between the DNA isolated from bladder cancer patients and the N-OH-AABP-modified DNA implicating the role of 4-ABP metabolites in the DNA damage and neo-antigenic epitope generation that could lead to the induction of antibodies in bladder cancer patients.

Hepatitis virus infection affects DNA methylation in mice with humanized livers.

PubMed

Okamoto, Yasuyuki; Shinjo, Keiko; Shimizu, Yasuhiro; Sano, Tsuyoshi; Yamao, Kenji; Gao, Wentao; Fujii, Makiko; Osada, Hirotaka; Sekido, Yoshitaka; Murakami, Shuko; Tanaka, Yasuhito; Joh, Takashi; Sato, Shinya; Takahashi, Satoru; Wakita, Takaji; Zhu, Jingde; Issa, Jean-Pierre J; Kondo, Yutaka

2014-02-01

Cells of tumors associated with chronic inflammation frequently have altered patterns of DNA methylation, including hepatocellular carcinomas. Chronic hepatitis has also been associated with aberrant DNA methylation, but little is known about their relationship. Pyrosequencing was used to determine the methylation status of cultured Huh7.5.1 hepatoma cells after hepatitis C virus (HCV) infection. We also studied mice with severe combined immunodeficiency carrying the urokinase-type plasminogen activator transgene controlled by an albumin promoter (urokinase-type plasminogen activator/severe combined immunodeficient mice), in which up to 85% of hepatocytes were replaced by human hepatocytes (chimeric mice). Mice were given intravenous injections of hepatitis B virus (HBV) or HCV, liver tissues were collected, and DNA methylation profiles were determined at different time points after infection. We also compared methylation patterns between paired samples of hepatocellular carcinomas and adjacent nontumor liver tissues from patients. No reproducible changes in DNA methylation were observed after infection of Huh7.5.1 cells with HCV. Livers from HBV- and HCV-infected mice had genome-wide, time-dependent changes in DNA methylation, compared with uninfected urokinase-type plasminogen activator/severe combined immunodeficient mice. There were changes in 160 ± 63 genes in HBV-infected and 237 ± 110 genes in HCV-infected mice. Methylation of 149 common genes increased in HBV- and HCV-infected mice; methylation of some of these genes also increased in hepatocellular carcinoma samples from patients compared with nontumor tissues. Expression of Ifng, which is expressed by natural killer cells, increased significantly in chimeric livers, in concordance with induction of DNA methylation, after infection with HBV or HCV. Induction of Ifng was reduced after administration of an inhibitor of natural killer cell function (anti-asialo GM1). In chimeric mice with humanized livers, infection with HBV and HCV appears to activate a natural kill cell-dependent innate immune response. This contributes to the induction and accumulation of aberrant DNA methylation in human hepatocytes. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Lactobacilli and bifidobacteria in human breast milk: influence of antibiotherapy and other host and clinical factors.

PubMed

Soto, Ana; Martín, Virginia; Jiménez, Esther; Mader, Isabelle; Rodríguez, Juan M; Fernández, Leonides

2014-07-01

The objective of this work was to study the lactobacilli and bifidobacteria population in human milk of healthy women, and to investigate the influence that several factors (including antibioteraphy during pregnancy and lactation, country and date of birth, delivery mode, or infant age) may exert on such population. A total of 160 women living in Germany or Austria provided the breast milk samples. Initially, 66 samples were randomly selected and cultured on MRS-Cys agar plates. Then, the presence of DNA from the genera Lactobacillus and Bifidobacterium, and from most of the Lactobacillus and Bifidobacterium species that were isolated, was assessed by qualitative polymerase chain reaction (PCR) using genus- and species-specific primers. Lactobacilli and bifidobacteria could be isolated from the milk of 27 (40.91%) and 7 (10.61%), respectively, of the 66 cultured samples. On the contrary, Lactobacillus and Bifidobacterium sequences were detected by PCR in 108 (67.50%) and 41 (25.62%), respectively, of the 160 samples analyzed. The Lactobacillus species most frequently isolated and detected was L salivarius (35.00%), followed by L fermentum (25.00%) and L gasseri (21.88%), whereas B breve (13.75%) was the bifidobacterial species most commonly recovered and whose DNA was most regularly found. The number of lactobacilli- or bifidobacteria-positive samples was significantly lower in women who had received antibiotherapy during pregnancy or lactation. Our results suggest that either the presence of lactobacilli and/or bifidobacteria or their DNA may constitute good markers of a healthy human milk microbiota that has not been altered by the use of antibiotics.

Towards establishing a human fecal contamination index in microbial source tracking

EPA Science Inventory

There have been significant advances in development of PCR-based methods to detect source associated DNA sequences (markers), but method evaluation has focused on performance with individual challenge samples. Little attention has been given to integration of multiple samples fro...

Joint Estimation of Contamination, Error and Demography for Nuclear DNA from Ancient Humans

PubMed Central

Slatkin, Montgomery

2016-01-01

When sequencing an ancient DNA sample from a hominin fossil, DNA from present-day humans involved in excavation and extraction will be sequenced along with the endogenous material. This type of contamination is problematic for downstream analyses as it will introduce a bias towards the population of the contaminating individual(s). Quantifying the extent of contamination is a crucial step as it allows researchers to account for possible biases that may arise in downstream genetic analyses. Here, we present an MCMC algorithm to co-estimate the contamination rate, sequencing error rate and demographic parameters—including drift times and admixture rates—for an ancient nuclear genome obtained from human remains, when the putative contaminating DNA comes from present-day humans. We assume we have a large panel representing the putative contaminant population (e.g. European, East Asian or African). The method is implemented in a C++ program called ‘Demographic Inference with Contamination and Error’ (DICE). We applied it to simulations and genome data from ancient Neanderthals and modern humans. With reasonable levels of genome sequence coverage (>3X), we find we can recover accurate estimates of all these parameters, even when the contamination rate is as high as 50%. PMID:27049965

Molecular Detection and Characterization of Tick-borne Pathogens in Dogs and Ticks from Nigeria

PubMed Central

Kamani, Joshua; Baneth, Gad; Mumcuoglu, Kosta Y.; Waziri, Ndadilnasiya E.; Eyal, Osnat; Guthmann, Yifat; Harrus, Shimon

2013-01-01

Background Only limited information is currently available on the prevalence of vector borne and zoonotic pathogens in dogs and ticks in Nigeria. The aim of this study was to use molecular techniques to detect and characterize vector borne pathogens in dogs and ticks from Nigeria. Methodology/Principal Findings Blood samples and ticks (Rhipicephalus sanguineus, Rhipicephalus turanicus and Heamaphysalis leachi) collected from 181 dogs from Nigeria were molecularly screened for human and animal vector-borne pathogens by PCR and sequencing. DNA of Hepatozoon canis (41.4%), Ehrlichia canis (12.7%), Rickettsia spp. (8.8%), Babesia rossi (6.6%), Anaplasma platys (6.6%), Babesia vogeli (0.6%) and Theileria sp. (0.6%) was detected in the blood samples. DNA of E. canis (23.7%), H. canis (21.1%), Rickettsia spp. (10.5%), Candidatus Neoehrlichia mikurensis (5.3%) and A. platys (1.9%) was detected in 258 ticks collected from 42 of the 181 dogs. Co- infections with two pathogens were present in 37% of the dogs examined and one dog was co-infected with 3 pathogens. DNA of Rickettsia conorii israelensis was detected in one dog and Rhipicephalus sanguineus tick. DNA of another human pathogen, Candidatus N. mikurensis was detected in Rhipicephalus sanguineus and Heamaphysalis leachi ticks, and is the first description of Candidatus N. mikurensis in Africa. The Theileria sp. DNA detected in a local dog in this study had 98% sequence identity to Theileria ovis from sheep. Conclusions/Significance The results of this study indicate that human and animal pathogens are abundant in dogs and their ticks in Nigeria and portray the potential high risk of human exposure to infection with these agents. PMID:23505591

Genomic Distribution and Inter-Sample Variation of Non-CpG Methylation across Human Cell Types

PubMed Central

Liao, Jing; Zhang, Yingying; Gu, Hongcang; Bock, Christoph; Boyle, Patrick; Epstein, Charles B.; Bernstein, Bradley E.; Lengauer, Thomas; Gnirke, Andreas; Meissner, Alexander

2011-01-01

DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three quarters of the total DNA methylation content in human and mouse cells. While the genomic distribution, inter-individual stability, and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpT, and CpC (non-CpG) dinucleotides. Here we report a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types. We confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Although no function has been assigned to it in pluripotency, our data highlight that non-CpG methylation patterns reappear upon iPS cell reprogramming. Intriguingly, the patterns are highly variable and show little conservation between different pluripotent cell lines. We find a strong correlation of non-CpG methylation and DNMT3 expression levels while showing statistical independence of non-CpG methylation from pluripotency associated gene expression. In line with these findings, we show that knockdown of DNMTA and DNMT3B in hESCs results in a global reduction of non-CpG methylation. Finally, non-CpG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of cytosine methylation patterns in human cells using a large representative sample set. PMID:22174693

Geographical and genospecies distribution of Borrelia burgdorferi sensu lato DNA detected in humans in the USA.

PubMed

Clark, Kerry L; Leydet, Brian F; Threlkeld, Clifford

2014-05-01

The present study investigated the cause of illness in human patients primarily in the southern USA with suspected Lyme disease based on erythema migrans-like skin lesions and/or symptoms consistent with early localized or late disseminated Lyme borreliosis. The study also included some patients from other states throughout the USA. Several PCR assays specific for either members of the genus Borrelia or only for Lyme group Borrelia spp. (Borrelia burgdorferi sensu lato), and DNA sequence analysis, were used to identify Borrelia spp. DNA in blood and skin biopsy samples from human patients. B. burgdorferi sensu lato DNA was found in both blood and skin biopsy samples from patients residing in the southern states and elsewhere in the USA, but no evidence of DNA from other Borrelia spp. was detected. Based on phylogenetic analysis of partial flagellin (flaB) gene sequences, strains that clustered separately with B. burgdorferi sensu stricto, Borrelia americana or Borrelia andersonii were associated with Lyme disease-like signs and symptoms in patients from the southern states, as well as from some other areas of the country. Strains most similar to B. burgdorferi sensu stricto and B. americana were found most commonly and appeared to be widely distributed among patients residing throughout the USA. The study findings suggest that human cases of Lyme disease in the southern USA may be more common than previously recognized and may also be caused by more than one species of B. burgdorferi sensu lato. This study provides further evidence that B. burgdorferi sensu stricto is not the only species associated with signs and/or symptoms consistent with Lyme borreliosis in the USA.

Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus.

PubMed

Ozga, Andrew T; Nieves-Colón, Maria A; Honap, Tanvi P; Sankaranarayanan, Krithivasan; Hofman, Courtney A; Milner, George R; Lewis, Cecil M; Stone, Anne C; Warinner, Christina

2016-06-01

Archaeological dental calculus is a rich source of host-associated biomolecules. Importantly, however, dental calculus is more accurately described as a calcified microbial biofilm than a host tissue. As such, concerns regarding destructive analysis of human remains may not apply as strongly to dental calculus, opening the possibility of obtaining human health and ancestry information from dental calculus in cases where destructive analysis of conventional skeletal remains is not permitted. Here we investigate the preservation of human mitochondrial DNA (mtDNA) in archaeological dental calculus and its potential for full mitochondrial genome (mitogenome) reconstruction in maternal lineage ancestry analysis. Extracted DNA from six individuals at the 700-year-old Norris Farms #36 cemetery in Illinois was enriched for mtDNA using in-solution capture techniques, followed by Illumina high-throughput sequencing. Full mitogenomes (7-34×) were successfully reconstructed from dental calculus for all six individuals, including three individuals who had previously tested negative for DNA preservation in bone using conventional PCR techniques. Mitochondrial haplogroup assignments were consistent with previously published findings, and additional comparative analysis of paired dental calculus and dentine from two individuals yielded equivalent haplotype results. All dental calculus samples exhibited damage patterns consistent with ancient DNA, and mitochondrial sequences were estimated to be 92-100% endogenous. DNA polymerase choice was found to impact error rates in downstream sequence analysis, but these effects can be mitigated by greater sequencing depth. Dental calculus is a viable alternative source of human DNA that can be used to reconstruct full mitogenomes from archaeological remains. Am J Phys Anthropol 160:220-228, 2016. © 2016 The Authors American Journal of Physical Anthropology Published by Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus

PubMed Central

Ozga, Andrew T.; Nieves‐Colón, Maria A.; Honap, Tanvi P.; Sankaranarayanan, Krithivasan; Hofman, Courtney A.; Milner, George R.; Lewis, Cecil M.; Stone, Anne C.

2016-01-01

ABSTRACT Objectives Archaeological dental calculus is a rich source of host‐associated biomolecules. Importantly, however, dental calculus is more accurately described as a calcified microbial biofilm than a host tissue. As such, concerns regarding destructive analysis of human remains may not apply as strongly to dental calculus, opening the possibility of obtaining human health and ancestry information from dental calculus in cases where destructive analysis of conventional skeletal remains is not permitted. Here we investigate the preservation of human mitochondrial DNA (mtDNA) in archaeological dental calculus and its potential for full mitochondrial genome (mitogenome) reconstruction in maternal lineage ancestry analysis. Materials and Methods Extracted DNA from six individuals at the 700‐year‐old Norris Farms #36 cemetery in Illinois was enriched for mtDNA using in‐solution capture techniques, followed by Illumina high‐throughput sequencing. Results Full mitogenomes (7–34×) were successfully reconstructed from dental calculus for all six individuals, including three individuals who had previously tested negative for DNA preservation in bone using conventional PCR techniques. Mitochondrial haplogroup assignments were consistent with previously published findings, and additional comparative analysis of paired dental calculus and dentine from two individuals yielded equivalent haplotype results. All dental calculus samples exhibited damage patterns consistent with ancient DNA, and mitochondrial sequences were estimated to be 92–100% endogenous. DNA polymerase choice was found to impact error rates in downstream sequence analysis, but these effects can be mitigated by greater sequencing depth. Discussion Dental calculus is a viable alternative source of human DNA that can be used to reconstruct full mitogenomes from archaeological remains. Am J Phys Anthropol 160:220–228, 2016. © 2016 The Authors American Journal of Physical Anthropology Published by Wiley Periodicals, Inc. PMID:26989998

The development of miniplex primer sets for the analysis of degraded DNA

NASA Astrophysics Data System (ADS)

McCord, Bruce; Opel, Kerry; Chung, Denise; Drabek, Jiri; Tatarek, Nancy; Meadows Jantz, Lee; Butler, John

2005-05-01

In this project, a new set of multiplexed PCR reactions has been developed for the analysis of degraded DNA. These DNA markers, known as Miniplexes, utilize primers that have shorter amplicons for use in short tandem repeat (STR) analysis of degraded DNA. In our work we have defined six of these new STR multiplexes, each of which consists of 3 to 4 reduced size STR loci, and each labeled with a different fluorescent dye. When compared to commercially available STR systems, reductions in size of up to 300 basepairs are possible. In addition, these newly designed amplicons consist of loci that are fully compatible with the the national computer DNA database known as CODIS. To demonstrate compatibility with commercial STR kits, a concordance study of 532 DNA samples of Caucasian, African American, and Hispanic origin was undertaken There was 99.77% concordance between allele calls with the two methods. Of these 532 samples, only 15 samples showed discrepancies at one of 12 loci. These occurred predominantly at 2 loci, vWA and D13S317. DNA sequencing revealed that these locations had deletions between the two primer binding sites. Uncommon deletions like these can be expected in certain samples and will not affect the utility of the Miniplexes as tools for degraded DNA analysis. The Miniplexes were also applied to enzymatically digested DNA to assess their potential in degraded DNA analysis. The results demonstrated a greatly improved efficiency in the analysis of degraded DNA when compared to commercial STR genotyping kits. A series of human skeletal remains that had been exposed to a variety of environmental conditions were also examined. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only sixteen percent of the samples tested generated full profiles with a commercial kit. In addition, complete profiles were obtained for eleven of the twelve Miniplex loci which had amplicon size ranges less than 200 base pairs. These data clearly demonstrate that smaller PCR amplicons provide an attractive alternative to mitochondrial DNA for forensic analysis of degraded DNA.

Estimates of Continental Ancestry Vary Widely among Individuals with the Same mtDNA Haplogroup

PubMed Central

Emery, Leslie S.; Magnaye, Kevin M.; Bigham, Abigail W.; Akey, Joshua M.; Bamshad, Michael J.

2015-01-01

The association between a geographical region and an mtDNA haplogroup(s) has provided the basis for using mtDNA haplogroups to infer an individual’s place of origin and genetic ancestry. Although it is well known that ancestry inferences using mtDNA haplogroups and those using genome-wide markers are frequently discrepant, little empirical information exists on the magnitude and scope of such discrepancies between multiple mtDNA haplogroups and worldwide populations. We compared genetic-ancestry inferences made by mtDNA-haplogroup membership to those made by autosomal SNPs in ∼940 samples of the Human Genome Diversity Panel and recently admixed populations from the 1000 Genomes Project. Continental-ancestry proportions often varied widely among individuals sharing the same mtDNA haplogroup. For only half of mtDNA haplogroups did the highest average continental-ancestry proportion match the highest continental-ancestry proportion of a majority of individuals with that haplogroup. Prediction of an individual’s mtDNA haplogroup from his or her continental-ancestry proportions was often incorrect. Collectively, these results indicate that for most individuals in the worldwide populations sampled, mtDNA-haplogroup membership provides limited information about either continental ancestry or continental region of origin. PMID:25620206

Toward Standardization of Epstein-Barr Virus DNA Load Monitoring: Unfractionated Whole Blood as Preferred Clinical Specimen

PubMed Central

Stevens, Servi J. C.; Pronk, Inge; Middeldorp, Jaap M.

2001-01-01

Epstein-Barr virus (EBV) DNA load monitoring in peripheral blood has been shown to be a useful tool for the diagnosis of aberrant EBV infections. In the present study we compared the relative diagnostic values of EBV DNA load monitoring in unfractionated whole blood and simultaneously obtained serum or plasma samples from Burkitt's lymphoma (BL) patients, transplant recipients, human immunodeficiency virus (HIV)-infected individuals, and infectious mononucleosis (IM) patients by a quantitative competitive PCR (Q-PCR). The EBV DNA load in BL patients was mainly situated in the cellular blood compartment (up to 4.5 × 106 copies/ml). EBV DNA loads in unfractionated whole blood and parallel serum samples showed no correlation. In transplant recipients, IM patients, and HIV-infected patients, the EBV burden in the circulation was almost exclusively restricted to the cellular blood compartment, because serum or plasma samples from these patients yielded negative results by Q-PCR, despite high viral loads in corresponding whole-blood samples. A 10-fold more sensitive but qualitative BamHI-W-repeat PCR occasionally revealed the presence of EBV at <2,000 copies of EBV DNA per ml of serum. Spiking of 100 copies of EBV DNA in samples with negative Q-PCR results excluded the presence of inhibitory factors in serum or plasma that influenced the Q-PCR result. Serum samples from all populations were often positive for β-globin DNA, indicating cell damage in vivo or during serum preparation. We conclude that serum is an undesirable clinical specimen for EBV DNA load monitoring because it omits the presence of cell-associated virus and uncontrolled cell lysis may give irreproducible results or overestimation of the DNA load. Unfractionated whole blood is strongly preferred since it combines all blood compartments that may harbor EBV and it best reflects the absolute viral burden in the patient's circulation. PMID:11283029

Enhanced DNA Profiling of the Semen Donor in Late Reported Sexual Assaults: Use of Y-Chromosome-Targeted Pre-amplification and Next Generation Y-STR Amplification Systems.

PubMed

Hanson, Erin K; Ballantyne, Jack

2016-01-01

In some cases of sexual assault the victim may not report the assault for several days after the incident due to various factors. The ability to obtain an autosomal STR profile of the semen donor from a living victim rapidly diminishes as the post-coital interval is extended due to the presence of only a small amount of male DNA amidst an overwhelming amount of female DNA. Previously, we have utilized various technological tools to overcome the limitations of male DNA profiling in extended interval post-coital samples including the use of Y-chromosome STR profiling, cervical sample, and post-PCR purification permitting the recovery of Y-STR profiles of the male DNA from samples collected 5-6 days after intercourse. Despite this success, the reproductive biology literature reports the presence of spermatozoa in the human cervix up to 7-10 days post-coitus. Therefore, novel and improved methods for recovery of male profiles in extended interval post-coital samples were required. Here, we describe enhanced strategies, including Y-chromosome-targeted pre-amplification and next generation Y-STR amplification kits, that have resulted in the ability to obtain probative male profiles from samples collected 6-9 days after intercourse.

Colorimetric determination of DNase I activity with a DNA-methyl green substrate.

PubMed

Sinicropi, D; Baker, D L; Prince, W S; Shiffer, K; Shak, S

1994-11-01

A simple, high throughput, and precise assay was developed for quantification of deoxyribonuclease I (DNase; IUB 3.1.21.1) activity. The method was adapted from the procedure devised by Kurnick which employs a substrate comprised of highly polymerized native DNA complexed with methyl green. Hydrolysis of the DNA produced unbound methyl green and a decrease in the absorbance of the solution at 620 nm. By adjusting the time and temperature of the reaction, the assay permits quantification of DNase activity over a wide concentration range (0.4 to 8900 ng/ml). Samples and standards were added to the substrate in microtiter plates and were incubated for 1-24 h at 25-37 degrees C to achieve the desired assay range. The DNase activity of the samples was interpolated from a standard curve generated with Pulmozyme recombinant human deoxyribonuclease I (rhDNase). Interassay precision was less than 12% CV and recovery was within 100 +/- 11%. Activity determination by the DNA-methyl green method correlated well with that determined by the widely used "hyperchromicity" method originated by Kunitz, which is based on the increase in absorbance at 260 nm upon hydrolysis of DNA. The DNA-methyl green assay was simpler and more versatile than the hyperchromicity method and was used to characterize the activity of rhDNase and DNase isolated from human urine.

Recovery of latent fingerprints and DNA on human skin.

PubMed

Färber, Doris; Seul, Andrea; Weisser, Hans-Joachim; Bohnert, Michael

2010-11-01

The project "Latent Fingerprints and DNA on Human Skin" was the first systematic research in Europe dealing with detection of fingerprints and DNA left by offenders on the skin of corpses. One thousand samples gave results that allow general statements on the materials and methods used. The tests were carried out according to a uniform trial structure. Fingerprints were deposited by natural donors on corpses. The latent fingerprints were treated with magnetic powder or black fingerprint powder. Afterward, they were lifted with silicone casting material (Isomark(®)) or gelatine foil. All lifts were swabbed to recover DNA. It was possible to visualize comparable and identifiable fingerprints on the skin of corpses (16%). In the same categories, magnetic powder (18.4%) yielded better results than black fingerprint powder (13.6%). The number of comparable and identifiable fingerprints decreased on the lifts (12.7%). Isomark(®) (14.9%) was the better lifting material in comparison with gelatine foil (10.1%). In one-third of the samples, DNA could be extracted from the powdered and lifted latents. Black fingerprint powder delivered the better result with a rate of 2.2% for full DNA profiles and profiles useful for exclusion in comparison with 1.8% for the magnetic powder traces. Isomark(®) (3.1%) yielded better results than gelatine foil (0.6%). © 2010 American Academy of Forensic Sciences.

Detection of Coxiella burnetii DNA on Small-Ruminant Farms during a Q Fever Outbreak in the Netherlands

PubMed Central

van der Plaats, R. Q. J.; de Heer, L.; Paauwe, R.; Schimmer, B.; Vellema, P.; van Rotterdam, B. J.; van Duynhoven, Y. T. H. P.

2012-01-01

During large Q fever outbreaks in the Netherlands between 2007 and 2010, dairy goat farms were implicated as the primary source of human Q fever. The transmission of Coxiella burnetii to humans is thought to occur primarily via aerosols, although available data on C. burnetii in aerosols and other environmental matrices are limited. During the outbreak of 2009, 19 dairy goat farms and one dairy sheep farm were selected nationwide to investigate the presence of C. burnetii DNA in vaginal swabs, manure, surface area swabs, milk unit filters, and aerosols. Four of these farms had a positive status during the Coxiella burnetii bulk milk monitoring program in 2009 and additionally reported abortion waves in 2008 or 2009. Eleven farms were reported as having positive bulk milk only, and five selected (control) farms had a bulk milk-negative status in 2009 and no reported Q fever history. Screening by quantitative PCR (qPCR) revealed that on farms with a history of abortions related to C. burnetii and, to a lesser extent, on farms positive by bulk milk monitoring, generally higher proportions of positive samples and higher levels of C. burnetii DNA within positive samples were observed than on the control farms. The relatively high levels of C. burnetii DNA in surface area swabs and aerosols sampled in stables of bulk milk-positive farms, including farms with a Q fever-related abortion history, support the hypothesis that these farms can pose a risk for the transmission of C. burnetii to humans. PMID:22247143

Phenotypes from ancient DNA: approaches, insights and prospects.

PubMed

Fortes, Gloria G; Speller, Camilla F; Hofreiter, Michael; King, Turi E

2013-08-01

The great majority of phenotypic characteristics are complex traits, complicating the identification of the genes underlying their expression. However, both methodological and theoretical progress in genome-wide association studies have resulted in a much better understanding of the underlying genetics of many phenotypic traits, including externally visible characteristics (EVCs) such as eye and hair color. Consequently, it has become possible to predict EVCs from human samples lacking phenotypic information. Predicting EVCs from genetic evidence is clearly appealing for forensic applications involving the personal identification of human remains. Now, a recent paper has reported the genetic determination of eye and hair color in samples up to 800 years old. The ability to predict EVCs from ancient human remains opens up promising perspectives for ancient DNA research, as this could allow studies to directly address archaeological and evolutionary questions related to the temporal and geographical origins of the genetic variants underlying phenotypes. © 2013 WILEY Periodicals, Inc.

Performance comparison of two commercial human whole-exome capture systems on formalin-fixed paraffin-embedded lung adenocarcinoma samples.

PubMed

Bonfiglio, Silvia; Vanni, Irene; Rossella, Valeria; Truini, Anna; Lazarevic, Dejan; Dal Bello, Maria Giovanna; Alama, Angela; Mora, Marco; Rijavec, Erika; Genova, Carlo; Cittaro, Davide; Grossi, Francesco; Coco, Simona

2016-08-30

Next Generation Sequencing (NGS) has become a valuable tool for molecular landscape characterization of cancer genomes, leading to a better understanding of tumor onset and progression, and opening new avenues in translational oncology. Formalin-fixed paraffin-embedded (FFPE) tissue is the method of choice for storage of clinical samples, however low quality of FFPE genomic DNA (gDNA) can limit its use for downstream applications. To investigate the FFPE specimen suitability for NGS analysis and to establish the performance of two solution-based exome capture technologies, we compared the whole-exome sequencing (WES) data of gDNA extracted from 5 fresh frozen (FF) and 5 matched FFPE lung adenocarcinoma tissues using: SeqCap EZ Human Exome v.3.0 (Roche NimbleGen) and SureSelect XT Human All Exon v.5 (Agilent Technologies). Sequencing metrics on Illumina HiSeq were optimal for both exome systems and comparable among FFPE and FF samples, with a slight increase of PCR duplicates in FFPE, mainly in Roche NimbleGen libraries. Comparison of single nucleotide variants (SNVs) between FFPE-FF pairs reached overlapping values >90 % in both systems. Both WES showed high concordance with target re-sequencing data by Ion PGM™ in 22 lung-cancer genes, regardless the source of samples. Exon coverage of 623 cancer-related genes revealed high coverage efficiency of both kits, proposing WES as a valid alternative to target re-sequencing. High-quality and reliable data can be successfully obtained from WES of FFPE samples starting from a relatively low amount of input gDNA, suggesting the inclusion of NGS-based tests into clinical contest. In conclusion, our analysis suggests that the WES approach could be extended to a translational research context as well as to the clinic (e.g. to study rare malignancies), where the simultaneous analysis of the whole coding region of the genome may help in the detection of cancer-linked variants.

A high-throughput Sanger strategy for human mitochondrial genome sequencing

PubMed Central

2013-01-01

Background A population reference database of complete human mitochondrial genome (mtGenome) sequences is needed to enable the use of mitochondrial DNA (mtDNA) coding region data in forensic casework applications. However, the development of entire mtGenome haplotypes to forensic data quality standards is difficult and laborious. A Sanger-based amplification and sequencing strategy that is designed for automated processing, yet routinely produces high quality sequences, is needed to facilitate high-volume production of these mtGenome data sets. Results We developed a robust 8-amplicon Sanger sequencing strategy that regularly produces complete, forensic-quality mtGenome haplotypes in the first pass of data generation. The protocol works equally well on samples representing diverse mtDNA haplogroups and DNA input quantities ranging from 50 pg to 1 ng, and can be applied to specimens of varying DNA quality. The complete workflow was specifically designed for implementation on robotic instrumentation, which increases throughput and reduces both the opportunities for error inherent to manual processing and the cost of generating full mtGenome sequences. Conclusions The described strategy will assist efforts to generate complete mtGenome haplotypes which meet the highest data quality expectations for forensic genetic and other applications. Additionally, high-quality data produced using this protocol can be used to assess mtDNA data developed using newer technologies and chemistries. Further, the amplification strategy can be used to enrich for mtDNA as a first step in sample preparation for targeted next-generation sequencing. PMID:24341507

Comparative sensitivity and inhibitor tolerance of GlobalFiler® PCR Amplification and Investigator® 24plex QS kits for challenging samples.

PubMed

Elwick, Kyleen; Mayes, Carrie; Hughes-Stamm, Sheree

2018-05-01

In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, been buried, decomposed, and/or contain inhibitory substances. This study compares the performance of a relatively new STR kit in the US market (Investigator® 24plex QS kit; Qiagen) with the GlobalFiler® PCR Amplification kit (Thermo Fisher Scientific) when genotyping highly inhibited and low level DNA samples. In this study, DNA samples ranging from 1 ng to 7.8 pg were amplified to define the sensitivity of two systems. In addition, DNA (1 ng and 0.1 ng input amounts) was spiked with various concentrations of five inhibitors common to human remains (humic acid, melanin, hematin, collagen, calcium). Furthermore, bone (N = 5) and tissue samples from decomposed human remains (N = 6) were used as mock casework samples for comparative analysis with both STR kits. The data suggest that the GlobalFiler® kit may be slightly more sensitive than the Investigator® kit. On average STR profiles appeared to be more balanced and average peak heights were higher when using the GlobalFiler® kit. However, the data also show that the Investigator® kit may be more tolerant to common PCR inhibitors. While both STR kits showed a decrease in alleles as the inhibitor concentration increased, more complete profiles were obtained when the Investigator® kit was used. Of the 11 bone and decomposed tissue samples tested, 8 resulted in more complete and balanced STR profiles when amplified with the GlobalFiler® kit. Copyright © 2018 Elsevier B.V. All rights reserved.

Prevalence of High risk Human Papillomavirus in cervical dysplasia and cancer samples from twin cities in Pakistan.

PubMed

Gul, Sana; Murad, Sheeba; Javed, Aneela

2015-05-01

Human Papilloma Virus (HPV) is small DNA virus mostly infecting mucosa and cutaneous keratinocytes. So far, more than 200 Human papillomaviruses are known. HPV have been divided into high- and low-risk on the basis of their oncogenic potential. High risk HPV is considered to be the main etiological cause for cervical cancer. The current study was designed to screen the local cervical cancer patients from the twin cities of Pakistan for the occurance of high risk HPV. A total of 67 formalin fixed paraffin-embedded samples of cervical cancer biopsies were obtained from the government hospitals in Islamabad and Rawalpindi. Cervical cancer biopsies were examined for the presence of HPV DNA. Polymerase chain reaction (PCR) was used for the amplification of a region in the HPV-L1 gene for the general detection of the Papilloma virus and for the genotype specific detection of high risk HPV 16 and 18 using the GP5/GP6 primers and genotype specific primers, respectively. HPV DNA was detected in 59 out of 67 samples analyzed. 30 samples showed the presence of HPV16 while 22 samples were positive for HPV18. HPV subtype could not be determined in 7 samples. Our results show a strong association between HPV infection and cervical cancer among women in twin cities of Pakistan. One way to minimize the disease burden in relation to HPV infection in Pakistani population is the use of prophylactic vaccines and routine screening. An early diagnosis of HPV infection will allow better health management to reduce the risk of developing cervical cancer. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Human papillomavirus DNA detection in plasma and cervical samples of women with a recent history of low grade or precancerous cervical dysplasia

PubMed Central

Sina, Federica; Piana, Andrea; Sotgiu, Giovanni; Dell’Anna, Tiziana; Musumeci, Rosario

2017-01-01

Circulating HPV DNA has been previously described in women with advanced stages of cervical cancer and has been suggested to be a prognostic marker of disease recurrences and metastases. Only a few studies have reported the presence of HPV DNA in bloodstream of patients with low grade or precancerous cervical lesions. This study aimed to define if HPV DNA could be detected in plasma samples of 120 women referred for a recent history of cervical dysplasia who presented with lesions ranging from High Squamous Intraepithelial Lesion (H-SIL) to regressed normal cytology. HPV DNA detection was carried out in both plasma and cervical samples using type-specific real-time quantitative PCR assays identifying oncogenic HPV 16, 18, 31, 33, 45, 51 and 52. Overall, 34.2% (41/120) of plasma samples were shown to be positive for HPV DNA detection; HPV 45 (46.3%), HPV-51 (29.6%), and HPV 16 (18.5%) were the most frequently identified genotypes. The rate of HPV detection in paired cervical and plasma samples increased with advancing disease stage, ranging from 15.4% in women with regressed lesions to 38.9% in women with HSIL; HPV 16 resulted the most common genotype identified in women found to be HPV DNA positive in both cervical and plasma samples. Moreover, HPV 16 showed the highest median viral load value in both cervical and plasma samples, with 48,313 copies/104 cells and 1,099 copies/ml, respectively. Results obtained in this study confirm that HPV DNA can be detected and quantified in plasma samples of women with asymptomatic cervical infection. Further knowledge on HPV dissemination through the blood stream of women with cervical lesions would be very important in better understanding the natural history of HPV infection as well as its potential role in other distant tumors. PMID:29182627

Human Parvovirus 4 in Nasal and Fecal Specimens from Children, Ghana

PubMed Central

Drexler, Jan Felix; Reber, Ulrike; Muth, Doreen; Herzog, Petra; Annan, Augustina; Ebach, Fabian; Sarpong, Nimarko; Acquah, Samuel; Adlkofer, Julia; Adu-Sarkodie, Yaw; Panning, Marcus; Tannich, Egbert; May, Jürgen; Drosten, Christian

2012-01-01

Nonparenteral transmission might contribute to human parvovirus 4 (PARV4) infections in sub-Saharan Africa. PARV4 DNA was detected in 8 (0.83%) of 961 nasal samples and 5 (0.53%) of 943 fecal samples from 1,904 children in Ghana. Virus concentrations ≤6–7 log10 copies/mL suggest respiratory or fecal–oral modes of PARV4 transmission. PMID:23018024

Using mitochondrial DNA to test the hypothesis of a European post-glacial human recolonization from the Franco-Cantabrian refuge.

PubMed

García, O; Fregel, R; Larruga, J M; Álvarez, V; Yurrebaso, I; Cabrera, V M; González, A M

2011-01-01

It has been proposed that the distribution patterns and coalescence ages found in Europeans for mitochondrial DNA (mtDNA) haplogroups V, H1 and H3 are the result of a post-glacial expansion from a Franco-Cantabrian refuge that recolonized central and northern areas. In contrast, in this refined mtDNA study of the Cantabrian Cornice that contributes 413 partial and 9 complete new mtDNA sequences, including a large Basque sample and a sample of Asturians, no experimental evidence was found to support the human refuge-expansion theory. In fact, all measures of gene diversity point to the Cantabrian Cornice in general and the Basques in particular, as less polymorphic for V, H1 and H3 than other southern regions in Iberia or in Central Europe. Genetic distances show the Cantabrian Cornice is a very heterogeneous region with significant local differences. The analysis of several minor subhaplogroups, based on complete sequences, also suggests different focal expansions over a local and peninsular range that did not affect continental Europe. Furthermore, all detected clinal trends show stronger longitudinal than latitudinal profiles. In Northern Iberia, it seems that the highest diversity values for some haplogroups with Mesolithic coalescence ages are centred on the Mediterranean side, including Catalonia and South-eastern France.

Polymerase chain reaction for detection of Leptospira spp. in clinical samples.

PubMed Central

Mérien, F; Amouriaux, P; Perolat, P; Baranton, G; Saint Girons, I

1992-01-01

A sensitive assay for Leptospira spp., the causative agent of leptospirosis, was developed on the basis of the polymerase chain reaction (PCR). A 331-bp sequence from the Leptospira interrogans serovar canicola rrs (16S) gene was amplified, and the PCR products were analyzed by DNA-DNA hybridization by using a 289-bp fragment internal to the amplified DNA. Specific PCR products also were obtained with DNA from the closely related nonpathogenic Leptospira biflexa but not with DNA from other spirochetes, such as Borrelia burgdorferi, Borrelia hermsii, Treponema denticola, Treponema pallidum, Spirochaeta aurantia, or more distant organisms such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, and Proteus mirabilis. The assay was able to detect as few as 10 bacteria. Leptospira DNA was detected in urine from experimentally infected mice. In addition, the test was found to be suitable for diagnosing leptospirosis in humans. Cerebrospinal fluid and urine from patients with leptospirosis were positive, whereas samples from control uninfected patients were negative. Images PMID:1400983

Investigating the Prevalence of Human Papilloma Virus in Squamous Cell Carcinoma of the Larynx and Its Correlation with Disease Prognosis

PubMed Central

Atighechi, Saeid; Meybodian, Mojtaba; Dadgarnia, Mohammad Hossein; Baradaranfar, Mohammad Hossein; Behniafard, Nasim

2016-01-01

Introduction: The human papilloma virus (HPV) can play a role in the development of head and neck squamous cell carcinoma (SCC). Our aim was to assess the prevalence of HPV DNA in SCC of the larynx. The impact of HPV infection on patient survival was also evaluated. Materials and Methods: This case-control study was performed in 44 patients with SCC of the larynx (case group), while the control group comprised samples obtained from cadavers with no previous history of malignancy. A preliminary pathologic evaluation was performed on all samples in the control group (36 samples) to ensure the absence of dysplasia or malignancy. Polymerase chain reaction (PCR) was used to detect HPV DNA. After completing the treatment protocol, patients were followed to assess the impact of HPV infection on overall survival (OS). Results: PCR evaluation in the case group showed that HPV DNA was successfully isolated from 11 (25%) samples, while only two (5.6%) HPV DNA-positive were obtained from cadavers. According to these results, a significant difference was obtained in the prevalence of HPV DNA and laryngeal SCC between cases and controls (P=0.031). No statistically significant difference was observed in the OS of patients with or without HPV infection in the case group (P=0.235). Conclusion: Based on these results, we suggest that the prevalence of HPV infection is higher in laryngeal SCC subjects compared with healthy individuals. Although a longer OS was seen in HPV-positive patients, survival analysis did not show a significant difference in the comparison of HPV-positive and negative findings in SCC patients. PMID:27429948

A large-scale dataset of single and mixed-source short tandem repeat profiles to inform human identification strategies: PROVEDIt.

PubMed

Alfonse, Lauren E; Garrett, Amanda D; Lun, Desmond S; Duffy, Ken R; Grgicak, Catherine M

2018-01-01

DNA-based human identity testing is conducted by comparison of PCR-amplified polymorphic Short Tandem Repeat (STR) motifs from a known source with the STR profiles obtained from uncertain sources. Samples such as those found at crime scenes often result in signal that is a composite of incomplete STR profiles from an unknown number of unknown contributors, making interpretation an arduous task. To facilitate advancement in STR interpretation challenges we provide over 25,000 multiplex STR profiles produced from one to five known individuals at target levels ranging from one to 160 copies of DNA. The data, generated under 144 laboratory conditions, are classified by total copy number and contributor proportions. For the 70% of samples that were synthetically compromised, we report the level of DNA damage using quantitative and end-point PCR. In addition, we characterize the complexity of the signal by exploring the number of detected alleles in each profile. Copyright © 2017 Elsevier B.V. All rights reserved.

PCR-based methods for identification of potentially zoonotic ascaridoid parasites of the dog, fox and cat.

PubMed

Jacobs, D E; Zhu, X; Gasser, R B; Chilton, N B

1997-11-01

Genomic DNA was extracted from ascaridoid nematodes collected from dogs, foxes and cats. A region spanning the second internal transcribed spacer (ITS-2) of the ribosomal DNA of each sample was amplified by PCR. Representative ITS-2 products for each nematode species (Toxocara canis, Toxocara cati and Toxascaris leonina) were sequenced. Restriction sites were identified for use as genetic markers in a PCR-linked RFLP assay. The three species could be differentiated from each other and from other ascaridoids that may be found in human tissues by use of two endonucleases, HinfI and RsaI. Primers were designed to unique regions of the ITS-2 sequences of the three species for use in diagnostic PCR procedures and primer sets evaluated against panels of homologous and heterologous DNA samples. Results suggest that both methods are good candidates for further development for the detection and/or identification of ascaridoid larvae in human tissues.

Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion.

PubMed

Kasurinen, Stefanie; Happo, Mikko S; Rönkkö, Teemu J; Orasche, Jürgen; Jokiniemi, Jorma; Kortelainen, Miika; Tissari, Jarkko; Zimmermann, Ralf; Hirvonen, Maija-Riitta; Jalava, Pasi I

2018-01-01

In vitro studies with monocultures of human alveolar cells shed deeper knowledge on the cellular mechanisms by which particulate matter (PM) causes toxicity, but cannot account for mitigating or aggravating effects of cell-cell interactions on PM toxicity. We assessed inflammation, oxidative stress as well as cytotoxic and genotoxic effects induced by PM from the combustion of different types of wood logs and softwood pellets in three cell culture setups: two monocultures of either human macrophage-like cells or human alveolar epithelial cells, and a co-culture of these two cell lines. The adverse effects of the PM samples were compared between these setups. We detected clear differences in the endpoints between the mono- and co-cultures. Inflammatory responses were more diverse in the macrophage monoculture and the co-culture compared to the epithelial cells where only an increase of IL-8 was detected. The production of reactive oxygen species was the highest in epithelial cells and macrophages seemed to have protective effects against oxidative stress from the PM samples. With no metabolically active cells at the highest doses, the cytotoxic effects of the PM samples from the wood log combustion were far more pronounced in the macrophages and the co-culture than in the epithelial cells. All samples caused DNA damage in macrophages, whereas only beech and spruce log combustion samples caused DNA damage in epithelial cells. The organic content of the samples was mainly associated with cytotoxicity and DNA damage, while the metal content of the samples correlated with the induction of inflammatory responses. All of the tested PM samples induce adverse effects and the chemical composition of the samples determines which pathway of toxicity is induced. In vitro testing of the toxicity of combustion-derived PM in monocultures of one cell line, however, is inadequate to account for all the possible pathways of toxicity.

Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion

PubMed Central

Happo, Mikko S.; Rönkkö, Teemu J.; Orasche, Jürgen; Jokiniemi, Jorma; Kortelainen, Miika; Tissari, Jarkko; Zimmermann, Ralf; Hirvonen, Maija-Riitta; Jalava, Pasi I.

2018-01-01

Background In vitro studies with monocultures of human alveolar cells shed deeper knowledge on the cellular mechanisms by which particulate matter (PM) causes toxicity, but cannot account for mitigating or aggravating effects of cell-cell interactions on PM toxicity. Methods We assessed inflammation, oxidative stress as well as cytotoxic and genotoxic effects induced by PM from the combustion of different types of wood logs and softwood pellets in three cell culture setups: two monocultures of either human macrophage-like cells or human alveolar epithelial cells, and a co-culture of these two cell lines. The adverse effects of the PM samples were compared between these setups. Results We detected clear differences in the endpoints between the mono- and co-cultures. Inflammatory responses were more diverse in the macrophage monoculture and the co-culture compared to the epithelial cells where only an increase of IL-8 was detected. The production of reactive oxygen species was the highest in epithelial cells and macrophages seemed to have protective effects against oxidative stress from the PM samples. With no metabolically active cells at the highest doses, the cytotoxic effects of the PM samples from the wood log combustion were far more pronounced in the macrophages and the co-culture than in the epithelial cells. All samples caused DNA damage in macrophages, whereas only beech and spruce log combustion samples caused DNA damage in epithelial cells. The organic content of the samples was mainly associated with cytotoxicity and DNA damage, while the metal content of the samples correlated with the induction of inflammatory responses. Conclusions All of the tested PM samples induce adverse effects and the chemical composition of the samples determines which pathway of toxicity is induced. In vitro testing of the toxicity of combustion-derived PM in monocultures of one cell line, however, is inadequate to account for all the possible pathways of toxicity. PMID:29466392

Incidence of human papilloma virus in esophageal squamous cell carcinoma in patients from the Lublin region.

PubMed

Dąbrowski, Andrzej; Kwaśniewski, Wojciech; Skoczylas, Tomasz; Bednarek, Wiesława; Kuźma, Dorota; Goździcka-Józefiak, Anna

2012-10-28

To assess the prevalence of human papilloma virus (HPV) in esophageal squamous cell carcinoma (ESCC) in the south-eastern region of Poland. The study population consisted of 56 ESCC patients and 35 controls. The controls were patients referred to our department due to other non-esophageal and non-oncological disorders with no gross or microscopic esophageal pathology as confirmed by endoscopy and histopathology. In the ESCC patients, samples were taken from normal mucosa (56 mucosa samples) and from the tumor (56 tumor samples). Tissue samples from the controls were taken from normal mucosa of the middle esophagus (35 control samples). Quantitative determination of DNA was carried out using a spectrophotometric method. Genomic DNA was isolated using the QIAamp DNA Midi Kit. HPV infection was identified following PCR amplification of the HPV gene sequence, using primers MY09 and MY11 complementary to the genome sequence of at least 33 types of HPV. The sequencing results were computationally analyzed using the basic local alignment search tool database. In tumor samples, HPV DNA was identified in 28 of 56 patients (50%). High risk HPV phenotypes (16 or/and 18) were found in 5 of 56 patients (8.9%), low risk in 19 of 56 patients (33.9%) and other types of HPV (37, 81, 97, CP6108) in 4 of 56 patients (7.1%). In mucosa samples, HPV DNA was isolated in 21 of 56 patients (37.5%). High risk HPV DNA was confirmed in 3 of 56 patients (5.3%), low risk HPV DNA in 12 of 56 patients (21.4%), and other types of HPV in 6 of 56 patients (10.7%). In control samples, HPV DNA was identified in 4 of 35 patients (11.4%) with no high risk HPV. The occurrence of HPV in ESCC patients was significantly higher than in the controls [28 of 56 (50%) vs 4 of 35 (11.4%), P < 0.001]. In esophageal cancer patients, both in tumor and mucosa samples, the predominant HPV phenotypes were low risk HPV, isolated 4 times more frequently than high risk phenotypes [19 of 56 (33.9%) vs 5 of 56 (8.9%), P < 0.001]. A higher prevalence of HPV was identified in female patients (71.4% vs 46.9%). Accordingly, the high risk phenotypes were isolated more frequently in female patients and this difference reached statistical significance [3 of 7 (42.9%) vs 2 of 49 (4.1%), P < 0.05]. Of the pathological characteristics, only an infiltrative pattern of macroscopic tumor type significantly correlated with the presence of HPV DNA in ESCC samples [20 of 27 (74.1%) vs 8 of 29 (27.6%) for ulcerative or protruding macroscopic type, P < 0.05]. The occurrence of total HPV DNA and both HPV high or low risk phenotypes did not significantly differ with regard to particular grades of cellular differentiation, phases in depth of tumor infiltration, grades of nodal involvement and stages of tumor progression. Low risk HPV phenotypes could be one of the co-activators or/and co-carcinogens in complex, progressive, multifactorial and multistep esophageal carcinogenesis.

Developmental validation of the DNAscan™ Rapid DNA Analysis™ instrument and expert system for reference sample processing.

PubMed

Della Manna, Angelo; Nye, Jeffrey V; Carney, Christopher; Hammons, Jennifer S; Mann, Michael; Al Shamali, Farida; Vallone, Peter M; Romsos, Erica L; Marne, Beth Ann; Tan, Eugene; Turingan, Rosemary S; Hogan, Catherine; Selden, Richard F; French, Julie L

2016-11-01

Since the implementation of forensic DNA typing in labs more than 20 years ago, the analysis procedures and data interpretation have always been conducted in a laboratory by highly trained and qualified scientific personnel. Rapid DNA technology has the potential to expand testing capabilities within forensic laboratories and to allow forensic STR analysis to be performed outside the physical boundaries of the traditional laboratory. The developmental validation of the DNAscan/ANDE Rapid DNA Analysis System was completed using a BioChipSet™ Cassette consumable designed for high DNA content samples, such as single source buccal swabs. A total of eight laboratories participated in the testing which totaled over 2300 swabs, and included nearly 1400 unique individuals. The goal of this extensive study was to obtain, document, analyze, and assess DNAscan and its internal Expert System to reliably genotype reference samples in a manner compliant with the FBI's Quality Assurance Standards (QAS) and the NDIS Operational Procedures. The DNAscan System provided high quality, concordant results for reference buccal swabs, including automated data analysis with an integrated Expert System. Seven external laboratories and NetBio, the developer of the technology, participated in the validation testing demonstrating the reproducibility and reliability of the system and its successful use in a variety of settings by numerous operators. The DNAscan System demonstrated limited cross reactivity with other species, was resilient in the presence of numerous inhibitors, and provided reproducible results for both buccal and purified DNA samples with sensitivity at a level appropriate for buccal swabs. The precision and resolution of the system met industry standards for detection of micro-variants and displayed single base resolution. PCR-based studies provided confidence that the system was robust and that the amplification reaction had been optimized to provide high quality results. The DNAscan integrated Expert System was examined as part of the Developmental Validation and successfully interpreted the over 2000 samples tested with over 99.998% concordant alleles. The system appropriately flagged samples for human review and failed both mixed samples and samples with insufficient genetic information. These results demonstrated the integrated Expert System makes correct allele calls without human intervention. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Detection and Genotyping of Human Papillomavirus in Self-Obtained Cervicovaginal Samples by Using the FTA Cartridge: New Possibilities for Cervical Cancer Screening ▿

PubMed Central

Lenselink, Charlotte H.; de Bie, Roosmarie P.; van Hamont, Dennis; Bakkers, Judith M. J. E.; Quint, Wim G. V.; Massuger, Leon F. A. G.; Bekkers, Ruud L. M.; Melchers, Willem J. G.

2009-01-01

This study assesses human papillomavirus (HPV) detection and genotyping in self-sampled genital smears applied to an indicating FTA elute cartridge (FTA cartridge). The study group consisted of 96 women, divided into two sample sets. All samples were analyzed by the HPV SPF10-Line Blot 25. Set 1 consisted of 45 women attending the gynecologist; all obtained a self-sampled cervicovaginal smear, which was applied to an FTA cartridge. HPV results were compared to a cervical smear (liquid based) taken by a trained physician. Set 2 consisted of 51 women who obtained a self-sampled cervicovaginal smear at home, which was applied to an FTA cartridge and to a liquid-based medium. DNA was obtained from the FTA cartridges by simple elution as well as extraction. Of all self-obtained samples of set 1, 62.2% tested HPV positive. The overall agreement between self- and physician-obtained samples was 93.3%, in favor of the self-obtained samples. In sample set 2, 25.5% tested HPV positive. The overall agreement for high-risk HPV presence between the FTA cartridge and liquid-based medium and between DNA elution and extraction was 100%. This study shows that HPV detection and genotyping in self-obtained cervicovaginal samples applied to an FTA cartridge is highly reliable. It shows a high level of overall agreement with HPV detection and genotyping in physician-obtained cervical smears and liquid-based self-samples. DNA can be obtained by simple elution and is therefore easy, cheap, and fast. Furthermore, the FTA cartridge is a convenient medium for collection and safe transport at ambient temperatures. Therefore, this method may contribute to a new way of cervical cancer screening. PMID:19553570

Detection and genotyping of human papillomavirus in self-obtained cervicovaginal samples by using the FTA cartridge: new possibilities for cervical cancer screening.

PubMed

Lenselink, Charlotte H; de Bie, Roosmarie P; van Hamont, Dennis; Bakkers, Judith M J E; Quint, Wim G V; Massuger, Leon F A G; Bekkers, Ruud L M; Melchers, Willem J G

2009-08-01

This study assesses human papillomavirus (HPV) detection and genotyping in self-sampled genital smears applied to an indicating FTA elute cartridge (FTA cartridge). The study group consisted of 96 women, divided into two sample sets. All samples were analyzed by the HPV SPF(10)-Line Blot 25. Set 1 consisted of 45 women attending the gynecologist; all obtained a self-sampled cervicovaginal smear, which was applied to an FTA cartridge. HPV results were compared to a cervical smear (liquid based) taken by a trained physician. Set 2 consisted of 51 women who obtained a self-sampled cervicovaginal smear at home, which was applied to an FTA cartridge and to a liquid-based medium. DNA was obtained from the FTA cartridges by simple elution as well as extraction. Of all self-obtained samples of set 1, 62.2% tested HPV positive. The overall agreement between self- and physician-obtained samples was 93.3%, in favor of the self-obtained samples. In sample set 2, 25.5% tested HPV positive. The overall agreement for high-risk HPV presence between the FTA cartridge and liquid-based medium and between DNA elution and extraction was 100%. This study shows that HPV detection and genotyping in self-obtained cervicovaginal samples applied to an FTA cartridge is highly reliable. It shows a high level of overall agreement with HPV detection and genotyping in physician-obtained cervical smears and liquid-based self-samples. DNA can be obtained by simple elution and is therefore easy, cheap, and fast. Furthermore, the FTA cartridge is a convenient medium for collection and safe transport at ambient temperatures. Therefore, this method may contribute to a new way of cervical cancer screening.

Prevalence and multiplicity of cutaneous beta papilloma viruses in plucked hairs depend on cellular DNA input.

PubMed

Weissenborn, S J; Neale, R; de Koning, M N C; Waterboer, T; Abeni, D; Bouwes Bavinck, J N; Wieland, U; Pfister, H J

2009-11-01

In view of the low loads of beta human papillomaviruses in skin samples, amounts of cellular DNA used in qualitative PCR may become limiting for virus detection and introduce variations in prevalence and multiplicity. This issue was explored within the context of a multicentre study and increasing prevalence and multiplicity was found with increasing input amounts of cellular DNA extracted from hair bulbs. To improve the quality and comparability between different epidemiologic studies ideally equal amounts of cellular DNA should be employed. When cellular DNA input varies this should be clearly taken into account in assessing viral prevalence and multiplicity.

Liquid nitrogen vapor is comparable to liquid nitrogen for storage of cryopreserved human sperm: evidence from the characteristics of post-thaw human sperm.

PubMed

Hu, Jingmei; Zhao, Shidou; Xu, Chengyan; Zhang, Lin; Lu, Shaoming; Cui, Linlin; Ma, Jinlong; Chen, Zi-Jiang

2015-11-01

To compare the differences in the characteristics of post-thaw human sperm after storage in either liquid nitrogen (LN2; -196 °C) or LN2 vapor (-167 °C). Experimental study. University hospital. Thirty healthy volunteers who agreed to donate their normal semen samples for infertility or research were included in the study. Semen samples (n = 30) were divided into eight aliquots and frozen. Four aliquots of each human semen sample were stored in LN2 (-196 °C), and the other four aliquots were stored in LN2 vapor (-167 °C). After 1, 3, 6, or 12 months, samples were thawed and analyzed. The motility was evaluated by the manual counting method. The viability was estimated by eosin staining. The morphology was analyzed by Diff-Quik staining. The sperm DNA integrity was determined with acridine orange fluorescent staining, and acrosin activity was assayed by the modified Kennedy method. The characteristics of post-thaw human sperm, including motility, viability, morphology, DNA integrity, and acrosin activity, showed no significant difference between LN2 and LN2 vapor storage for the different time periods. LN2 vapor was comparable to LN2 in post-thaw sperm characteristics, suggesting that LN2 vapor may be substituted for LN2 for the long-term storage of human sperm. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Hamburger polyomaviruses

PubMed Central

Peretti, Alberto; FitzGerald, Peter C.; Bliskovsky, Valery

2015-01-01

Epidemiological studies have suggested that consumption of beef may correlate with an increased risk of colorectal cancer. One hypothesis to explain this proposed link might be the presence of a carcinogenic infectious agent capable of withstanding cooking. Polyomaviruses are a ubiquitous family of thermostable non-enveloped DNA viruses that are known to be carcinogenic. Using virion enrichment, rolling circle amplification (RCA) and next-generation sequencing, we searched for polyomaviruses in meat samples purchased from several supermarkets. Ground beef samples were found to contain three polyomavirus species. One species, bovine polyomavirus 1 (BoPyV1), was originally discovered as a contaminant in laboratory FCS. A previously unknown species, BoPyV2, occupies the same clade as human Merkel cell polyomavirus and raccoon polyomavirus, both of which are carcinogenic in their native hosts. A third species, BoPyV3, is related to human polyomaviruses 6 and 7. Examples of additional DNA virus families, including herpesviruses, adenoviruses, circoviruses and gyroviruses were also detected either in ground beef samples or in comparison samples of ground pork and ground chicken. The results suggest that the virion enrichment/RCA approach is suitable for random detection of essentially any DNA virus with a detergent-stable capsid. It will be important for future studies to address the possibility that animal viruses commonly found in food might be associated with disease. PMID:25568187

Season of conception in rural gambia affects DNA methylation at putative human metastable epialleles.

PubMed

Waterland, Robert A; Kellermayer, Richard; Laritsky, Eleonora; Rayco-Solon, Pura; Harris, R Alan; Travisano, Michael; Zhang, Wenjuan; Torskaya, Maria S; Zhang, Jiexin; Shen, Lanlan; Manary, Mark J; Prentice, Andrew M

2010-12-23

Throughout most of the mammalian genome, genetically regulated developmental programming establishes diverse yet predictable epigenetic states across differentiated cells and tissues. At metastable epialleles (MEs), conversely, epigenotype is established stochastically in the early embryo then maintained in differentiated lineages, resulting in dramatic and systemic interindividual variation in epigenetic regulation. In the mouse, maternal nutrition affects this process, with permanent phenotypic consequences for the offspring. MEs have not previously been identified in humans. Here, using an innovative 2-tissue parallel epigenomic screen, we identified putative MEs in the human genome. In autopsy samples, we showed that DNA methylation at these loci is highly correlated across tissues representing all 3 embryonic germ layer lineages. Monozygotic twin pairs exhibited substantial discordance in DNA methylation at these loci, suggesting that their epigenetic state is established stochastically. We then tested for persistent epigenetic effects of periconceptional nutrition in rural Gambians, who experience dramatic seasonal fluctuations in nutritional status. DNA methylation at MEs was elevated in individuals conceived during the nutritionally challenged rainy season, providing the first evidence of a permanent, systemic effect of periconceptional environment on human epigenotype. At MEs, epigenetic regulation in internal organs and tissues varies among individuals and can be deduced from peripheral blood DNA. MEs should therefore facilitate an improved understanding of the role of interindividual epigenetic variation in human disease.

Quantification of tamoxifen DNA adducts using on-line sample preparation and HPLC-electrospray ionization tandem mass spectrometry.

PubMed

Gamboa da Costa, Gonçalo; Marques, M Matilde; Beland, Frederick A; Freeman, James P; Churchwell, Mona I; Doerge, Daniel R

2003-03-01

The nonsteroidal antiestrogen tamoxifen is used as an adjuvant chemotherapeutic agent for the treatment of all stages of hormone-dependent breast cancer and more recently as a chemopreventive agent in women with elevated risk of developing the disease. While clearly beneficial for the treatment of breast cancer, tamoxifen has been reported to increase the risk of endometrial cancer in women. Furthermore, it has been shown to be hepatocarcinogenic in rats. Tamoxifen is clearly genotoxic in rat liver, as indicated by the formation of DNA adducts; the occurrence of tamoxifen DNA adducts in human endometrial tissue is more controversial. The detection and quantitation of tamoxifen DNA adducts have relied primarily upon (32)P-postlabeling, with other techniques, such as immunoassays and accelerator mass spectrometry, being used to a much lesser extent. To expand the range of available analytical methodologies for quantifying tamoxifen DNA adducts, we have developed an assay using on-line sample preparation, coupled with HPLC and electrospray ionization tandem mass spectrometry (ES-MS/MS). alpha-Acetoxytamoxifen was reacted with salmon testis DNA at ratios between 0.1 ng and 1 mg alpha-acetoxytamoxifen per mg DNA. After enzymatic hydrolysis to nucleosides, the most highly modified DNA samples were analyzed by HPLC-UV, which indicated the presence of two adduct peaks in approximately a 1:4 ratio. The major adduct was isolated, rigorously characterized as (E)-alpha-(deoxyguanosin-N(2)-yl)tamoxifen, and quantified on the basis of its molar extinction coefficient. A similar reaction was conducted with [N(CD(3))(2)]-alpha-acetoxytamoxifen to prepare a deuterated adduct that could serve as an internal standard for ES-MS/MS. The limit of detection for the HPLC-ES-MS/MS method was approximately 5 adducts/10(9) nucleotides, with an intra- and interassay precision of 3% relative standard deviation. The method was validated over the range of 8-1 520,000 adducts/10(8) nucleotides using 100 microg samples of DNA modified in vitro. Analysis of liver DNA from female Sprague-Dawley rats treated by gavage with seven daily doses of 20 mg tamoxifen/kg body weight gave a value of 496 +/- 16 adducts/10(8) nucleotides for (E)-alpha-(deoxyguanosin-N(2)-yl)tamoxifen and 626 +/- 18 adducts/10(8) nucleotides for (E)-alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen. These data indicate that the HPLC-ES-MS/MS methodology has sufficient sensitivity and precision to be useful in the analysis of tamoxifen DNA adducts formed in vivo in experimental models and may be able to detect tamoxifen DNA adduct formation in human tissue samples.

Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malik, Afshan N., E-mail: afshan.malik@kcl.ac.uk; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana

2011-08-19

Highlights: {yields} Mitochondrial dysfunction is central to many diseases of oxidative stress. {yields} 95% of the mitochondrial genome is duplicated in the nuclear genome. {yields} Dilution of untreated genomic DNA leads to dilution bias. {yields} Unique primers and template pretreatment are needed to accurately measure mitochondrial DNA content. -- Abstract: Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that themore » methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as {beta}-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a 'dilution bias' when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.« less

The response of single human cells to zero-gravity

NASA Technical Reports Server (NTRS)

Montgomery, P. O., Jr.; Cook, J. E.; Reynolds, R. C.; Paul, J. S.; Hayflick, L.; Stock, D.; Shulz, W. W.; Kimzey, S. L.; Thirolf, R. G.; Rogers, T.

1977-01-01

Microscopic and histochemical evaluations of human embrionic lung cells after exposure to zero-gravity are reported. Growth curves, DNA microspectrophotometry, phase microscopy, and ultrastructural studies of fixed cells revealed no effects on the cultures. Minor unexplained differences have been found in biochemical constituents of the samples.

Estimating HPV DNA Deposition Between Sexual Partners Using HPV Concordance, Y Chromosome DNA Detection, and Self-reported Sexual Behaviors.

PubMed

Malagón, Talía; Burchell, Ann N; El-Zein, Mariam; Guénoun, Julie; Tellier, Pierre-Paul; Coutlée, François; Franco, Eduardo L

2017-12-05

Detection of human papillomavirus (HPV) DNA in genital samples may not always represent true infections but may be depositions from infected sexual partners. We examined whether sexual risk factors and a biomarker (Y chromosome DNA) were associated with genital HPV partner concordance and estimated the fraction of HPV detections potentially attributable to partner deposition. The HITCH study enrolled young women attending a university or college in Montréal, Canada, and their male partners, from 2005 to 2010. We tested baseline genital samples for Y chromosome DNA and HPV DNA using polymerase chain reaction. Type-specific HPV concordance was 42.4% in partnerships where at least one partner was HPV DNA positive. Y chromosome DNA predicted type-specific HPV concordance in univariate analyses, but in multivariable models the independent predictors of concordance were days since last vaginal sex (26.5% higher concordance 0-1 vs 8-14 days after last vaginal sex) and condom use (22.6% higher concordance in never vs always users). We estimated that 14.1% (95% confidence interval [CI], 6.3-21.9%) of HPV DNA detections in genital samples were attributable to vaginal sex in the past week. A substantial proportion of HPV DNA detections may be depositions due to recent unprotected vaginal sex. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Fast Technology Analysis (FTA) Enables Identification of Species and Genotypes of Latent Microsporidia Infections in Healthy Native Cameroonians

PubMed Central

Ndzi, Edward S.; Asonganyi, Tazoacha; Nkinin, Mary Bello; Xiao, Lihua; Didier, Elizabeth S.; Bowers, Lisa C.; Nkinin, Stephenson W.; Kaneshiro, Edna S.

2015-01-01

Several enteric microsporidia species have been detected in humans and other vertebrates and their identifications at the genotype level are currently being elucidated. As advanced methods, reagents, and disposal kits for detecting and identifying pathogens become commercially available, it is important to test them in settings other than in laboratories with “state-of-the-art” equipment and well-trained staff members. In the present study, we sought to detect microsporidia DNA preserved and extracted from FTA (fast technology analysis) cards spotted with human fecal suspensions obtained from Cameroonian volunteers living in the capital city of Yaoundé to preclude the need for employing spore-concentrating protocols. Further, we tested whether amplicon nucleotide sequencing approaches could be used on small aliquots taken from the cards to elucidate the diversity of microsporidia species and strains infecting native residents. Of 196 samples analyzed, 12 (6.1%) were positive for microsporidia DNA; Enterocytozoon bieneusi (Type IV and KIN-1), Encephalitozoon cuniculi, and Encephalitozoon intestinalis were identified. These data demonstrate the utility of the FTA cards in identifying genotypes of microsporidia DNA in human fecal samples that may be applied to field testing for prevalence studies. PMID:26303263

High expression of DNA methyltransferases in primary human medulloblastoma.

PubMed

Pócza, T; Krenács, T; Turányi, E; Csáthy, J; Jakab, Z; Hauser, P

2016-01-01

Epigenetic alterations have been implicated in cancer development. DNA methylation modulates gene expression, which is catalyzed by DNA methyltransferases (DNMTs). The objective of our study was to evaluate expression of DNMTs in medulloblastoma and analyze its correlation with clinical features. Nuclear expression of DNMT1, DNMT3A and DNMT3B was analyzed in human primary medulloblastoma of 44 patients using immunohistochemistry. Correlation of expression of DNMT levels with classical histological subtypes, novel molecular subgroups and survival of patients was analyzed. Elevated expression of DNMT1, DNMT3A and DNMT3B was observed in 63.64%, 68.18% and 72.73% of all cases, respectively. None of them showed a correlation with classical histology or survival. Concerning molecular subtypes, significantly higher expression of DNMT1 was observed in the SHH group compared to non-SHH samples (p = 0.02), but without significant difference in DNMT3A or DNMT3B levels between any subtypes. In conclusion, DNMT1, DNMT3A and DNMT3B are highly expressed in human medulloblastoma samples, suggesting that promoter hypermethylation may play a role in medulloblastoma development. Demethylation of tumor suppressor gene promoters may be considered as a possible future target in therapy of medulloblastoma.

Prevalence of human papilloma virus in marginal periodontium and its association with periodontitis: A cross sectional study.

PubMed

Jacob, Anila; Janam, Presanthila; Babu Vijayamma, Janki Mohan

2014-07-01

Bacterial pathogens in dental plaque are necessary for the development of periodontitis but this etiology alone does not explain all its clinicopathologic features. Researchers have proven the role of certain viruses like herpes virus in periodontal disease which implies that other viral agents like human papilloma virus may also be involved. This cross-sectional study was conducted to determine the proportion of patients with human papilloma virus (HPV-16) in marginal periodontium by analyzing DNA from the gingival tissue sample and to understand its association with periodontitis. 102 systemically healthy patients between the age group of 15 and 70 years reporting to the Department of Periodontology who required surgical intervention (flap surgery for patients with periodontitis and crown lengthening for healthy patients) with internal bevel gingivectomy were selected. After scaling and root planning, gingival tissue was collected during the respective surgical procedure. DNA was isolated and amplified using specific primers for HPV-16 by polymerase chain reaction (PCR). The amplified products were checked by agarose gel electrophoresis. No HPV DNA was detected in the 102 samples analyzed. Marginal periodontium does not contain HPV in this study population and hence there was no association between HPV and periodontitis.

In situ Localization of the Human Multidrug‐resistance Gene mRNA Using Thymine‐Thymine Dimerized Single‐stranded cDNA

PubMed Central

Koji, Takehiko; Ueda, Kazumitsu; Pastan, Ira; Gottesman, Michael M.; Nakane, Paul K.; Mori, Shigeo

1990-01-01

In order to detect the mRNA transcribed from the multidrug‐resistance gene (MDR1), thymine‐thymine (T‐T) dimerized single‐stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues. S1 nuclease digestion rather than sonication was used to obtain short T‐T dimerized single‐stranded DNA (300–400 bases) so that they could penetrate well into the cytoplasm. The hybridized T‐T DNA was detected immunohisto‐chemically using rabbit anti‐T‐T DNA antibody (Ab) and peroxidase‐labeled goat anti‐rabbit IgG Ab. Employing this system, MDR1 mRNA could be localized clearly in the human multidrug‐resistant cell lines K562/ADM, CEM/VLB, 2780ad, and KBC4 cells as well as in human fetal kidney and gastric carcinoma. Furthermore, our system successfully detected the expression of MDR1 mRNA in cell lines of increasing resistance. These results paralleled results obtained at the protein level by immunohistochemistry. The analysis of MDR1 RNA expression by this in situ hybridization technique should be useful in the study of normal human tissues and tumor samples expressing the MDR1 gene. PMID:1977730

An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

PubMed Central

Sterling, Catherine H.; Veksler-Lublinsky, Isana; Ambros, Victor

2015-01-01

The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids. PMID:25056322

Bioethical Biobanks: Three Concerns in Designing and Using Law Enforcement DNA Identification Databases

DOE Office of Scientific and Technical Information (OSTI.GOV)

D.H. Kaye

2006-10-19

Federal and state law enforcement authorities have amassed large collections of DNA samples and the identifying profiles derived from them. These databases help to identify the guilty and to exonerate the innocent, but as the databanks grow, so do fears about civil liberties. The research reported here discusses three legal and social policy issues that have been raised in regard to these biobanks—the choice of loci to type for identifying individuals, the indefinite retention of DNA samples, and the use of the DNA samples or the identifying profiles for research purposes. It also considers the possible value of the databasesmore » for research into the genetics of human behavior and the ethics of using them for this purpose. It rejects the broad claim that such research is inherently unethical but proposes procedures for ensuring that the value of the proposed research justifies any psychosocial or other risks to the subjects of the research.« less

Mitochondrial DNA variant at HVI region as a candidate of genetic markers of type 2 diabetes

NASA Astrophysics Data System (ADS)

Gumilar, Gun Gun; Purnamasari, Yunita; Setiadi, Rahmat

2016-02-01

Mitochondrial DNA (mtDNA) is maternally inherited. mtDNA mutations which can contribute to the excess of maternal inheritance of type 2 diabetes. Due to the high mutation rate, one of the areas in the mtDNA that is often associated with the disease is the hypervariable region I (HVI). Therefore, this study was conducted to determine the genetic variants of human mtDNA HVI that related to the type 2 diabetes in four samples that were taken from four generations in one lineage. Steps being taken include the lyses of hair follicles, amplification of mtDNA HVI fragment using Polymerase Chain Reaction (PCR), detection of PCR products through agarose gel electrophoresis technique, the measurement of the concentration of mtDNA using UV-Vis spectrophotometer, determination of the nucleotide sequence via direct sequencing method and analysis of the sequencing results using SeqMan DNASTAR program. Based on the comparison between nucleotide sequence of samples and revised Cambridge Reference Sequence (rCRS) obtained six same mutations that these are C16147T, T16189C, C16193del, T16127C, A16235G, and A16293C. After comparing the data obtained to the secondary data from Mitomap and NCBI, it were found that two mutations, T16189C and T16217C, become candidates as genetic markers of type 2 diabetes even the mutations were found also in the generations of undiagnosed type 2 diabetes. The results of this study are expected to give contribution to the collection of human mtDNA database of genetic variants that associated to metabolic diseases, so that in the future it can be utilized in various fields, especially in medicine.

Fellow travellers: a concordance of colonization patterns between mice and men in the North Atlantic region

PubMed Central

2012-01-01

Background House mice (Mus musculus) are commensals of humans and therefore their phylogeography can reflect human colonization and settlement patterns. Previous studies have linked the distribution of house mouse mitochondrial (mt) DNA clades to areas formerly occupied by the Norwegian Vikings in Norway and the British Isles. Norwegian Viking activity also extended further westwards in the North Atlantic with the settlement of Iceland, short-lived colonies in Greenland and a fleeting colony in Newfoundland in 1000 AD. Here we investigate whether house mouse mtDNA sequences reflect human history in these other regions as well. Results House mice samples from Iceland, whether from archaeological Viking Age material or from modern-day specimens, had an identical mtDNA haplotype to the clade previously linked with Norwegian Vikings. From mtDNA and microsatellite data, the modern-day Icelandic mice also share the low genetic diversity shown by their human hosts on Iceland. Viking Age mice from Greenland had an mtDNA haplotype deriving from the Icelandic haplotype, but the modern-day Greenlandic mice belong to an entirely different mtDNA clade. We found no genetic association between modern Newfoundland mice and the Icelandic/ancient Greenlandic mice (no ancient Newfoundland mice were available). The modern day Icelandic and Newfoundland mice belong to the subspecies M. m. domesticus, the Greenlandic mice to M. m. musculus. Conclusions In the North Atlantic region, human settlement history over a thousand years is reflected remarkably by the mtDNA phylogeny of house mice. In Iceland, the mtDNA data show the arrival and continuity of the house mouse population to the present day, while in Greenland the data suggest the arrival, subsequent extinction and recolonization of house mice - in both places mirroring the history of the European human host populations. If house mice arrived in Newfoundland with the Viking settlers at all, then, like the humans, their presence was also fleeting and left no genetic trace. The continuity of mtDNA haplotype in Iceland over 1000 years illustrates that mtDNA can retain the signature of the ancestral house mouse founders. We also show that, in terms of genetic variability, house mouse populations may also track their host human populations. PMID:22429664

Human Papillomavirus DNA Detection in Menstrual Blood from Patients with Cervical Intraepithelial Neoplasia and Condyloma Acuminatum ▿

PubMed Central

Wong, Sze Chuen Cesar; Au, Thomas Chi Chuen; Chan, Sammy Chung Sum; Chan, Charles Ming Lok; Lam, Money Yan Yee; Zee, Benny Chung Ying; Pong, Wei Mei; Chan, Anthony Tak Cheung

2010-01-01

The Papanicolaou test generates pain and embarrassment, and cytology screening has limited sensitivity for detection of cervical neoplasia. These factors urge the use of another screening test that can overcome these limitations. We explore a completely noninvasive method using detection of human papillomavirus (HPV) DNA in women's menstrual blood (MB). The participants were divided into 3 cohorts: (i) 235 patients with cervical intraepithelial neoplasia 3 (CIN 3) (n = 48), CIN 2 (n = 60), CIN 1 (n = 58), or condyloma acuminatum (CAC) (n = 69) before treatment or remission; (ii) from the first cohort of patients, 108 CIN 3 or CIN 2 patients after treatment and 62 CIN 1 or CAC patients after remission; and (iii) 323 apparently normal subjects (ANS) without any cervical disease. The HPV genotypes of the infected patients were confirmed by direct sequencing. Quantitative real-time PCR (QRT-PCR) was used to measure the MB HPV16 load for 15 infected patients. Results showed that the sensitivity, specificity, and positive and negative predictive values for detection of MB HPV DNA in samples from patients with CIN or CAC were 82.8%, 93.1%, 90.0%, and 87.9%, respectively. Moreover, MB HPV DNA was found in samples from 22.2% of CIN 3 or CIN 2 patients after treatment, 0.0% of CIN 1 or CAC patients after remission, and 8.1% of ANS, 4 of whom were found to have CIN 1 or CAC. Furthermore, QRT-PCR showed that the normalized MB HPV16 DNA copy numbers in samples from patients with CIN 1 to CIN 3 were significantly increased. These preliminary results suggested that MB HPV DNA is a potential noninvasive marker for these premalignant cervical diseases. PMID:20089764

Human papillomavirus DNA detection in menstrual blood from patients with cervical intraepithelial neoplasia and condyloma acuminatum.

PubMed

Wong, Sze Chuen Cesar; Au, Thomas Chi Chuen; Chan, Sammy Chung Sum; Chan, Charles Ming Lok; Lam, Money Yan Yee; Zee, Benny Chung Ying; Pong, Wei Mei; Chan, Anthony Tak Cheung

2010-03-01

The Papanicolaou test generates pain and embarrassment, and cytology screening has limited sensitivity for detection of cervical neoplasia. These factors urge the use of another screening test that can overcome these limitations. We explore a completely noninvasive method using detection of human papillomavirus (HPV) DNA in women's menstrual blood (MB). The participants were divided into 3 cohorts: (i) 235 patients with cervical intraepithelial neoplasia 3 (CIN 3) (n = 48), CIN 2 (n = 60), CIN 1 (n = 58), or condyloma acuminatum (CAC) (n = 69) before treatment or remission; (ii) from the first cohort of patients, 108 CIN 3 or CIN 2 patients after treatment and 62 CIN 1 or CAC patients after remission; and (iii) 323 apparently normal subjects (ANS) without any cervical disease. The HPV genotypes of the infected patients were confirmed by direct sequencing. Quantitative real-time PCR (QRT-PCR) was used to measure the MB HPV16 load for 15 infected patients. Results showed that the sensitivity, specificity, and positive and negative predictive values for detection of MB HPV DNA in samples from patients with CIN or CAC were 82.8%, 93.1%, 90.0%, and 87.9%, respectively. Moreover, MB HPV DNA was found in samples from 22.2% of CIN 3 or CIN 2 patients after treatment, 0.0% of CIN 1 or CAC patients after remission, and 8.1% of ANS, 4 of whom were found to have CIN 1 or CAC. Furthermore, QRT-PCR showed that the normalized MB HPV16 DNA copy numbers in samples from patients with CIN 1 to CIN 3 were significantly increased. These preliminary results suggested that MB HPV DNA is a potential noninvasive marker for these premalignant cervical diseases.

Glossary

MedlinePlus

... array, and oligo/SNP combination array. Related terms: comparative genomic hybridization ; copy number variant ; SNP array chromosome ... for example, the AB blood groups in humans comparative genomic hybridization Method in which two DNA samples ( ...

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation.

PubMed

Tong, Yu; Shen, Shizhen; Jiang, Hui; Chen, Zhi

2017-01-01

Gene mutation has been considered a research hotspot, and the rapid development of biomedicine has enabled significant advances in the evaluation of gene mutations. The advent of digital polymerase chain reaction (dPCR) elevates the detection of gene mutations to unprecedented levels of precision, especially in cancer-associated genes. dPCR has been utilized in the detection of tumor markers in cell-free DNA (cfDNA) samples from patients with different types of cancer in samples such as plasma, cerebrospinal fluid, urine and sputum, which confers significant value for dPCR in both clinical applications and basic research. Moreover, dPCR is extensively used in detecting pathogen mutations related to typical features of infectious diseases (e.g., drug resistance) and mutation status of heteroplasmic mitochondrial DNA, which determines the manifestation and progression of mtDNA-related diseases, as well as allows for the prenatal diagnosis of monogenic diseases and the assessment of the genome editing effects. Compared with real-time PCR (qPCR) and sequencing, the higher sensitivity and accuracy of dPCR indicates a great advantage in the detection of rare mutation. As a new technique, dPCR has some limitations, such as the necessity of highly allele-specific probes and a large sample volume. In this review, we summarize the application of dPCR in the detection of human disease-associated gene mutations. © 2017 The Author(s). Published by S. Karger AG, Basel.

PEGylated Polyaniline Nanofibers: Antifouling and Conducting Biomaterial for Electrochemical DNA Sensing.

PubMed

Hui, Ni; Sun, Xiaotian; Niu, Shuyan; Luo, Xiliang

2017-01-25

Biofouling arising from nonspecific adsorption is a substantial outstanding challenge in diagnostics and disease monitoring, and antifouling sensing interfaces capable of reducing the nonspecific adsorption of proteins from biological complex samples are highly desirable. We present herein the preparation of novel composite nanofibers through the grafting of polyethylene glycol (PEG) polymer onto polyaniline (PANI) nanofibers and their application in the development of antifouling electrochemical biosensors. The PEGylated PANI (PANI/PEG) nanofibers possessed large surface area and remained conductive and at the same time demonstrated excellent antifouling performances in single protein solutions as well as complex human serum samples. Sensitive and low fouling electrochemical biosensors for the breast cancer susceptibility gene (BRCA1) can be easily fabricated through the attachment of DNA probes to the PANI/PEG nanofibers. The biosensor showed a very high sensitivity to target BRCA1 with a linear range from 0.01 pM to 1 nM and was also efficient enough to detect DNA mismatches with satisfactory selectivity. Moreover, the DNA biosensor based on the PEGylated PANI nanofibers supported the quantification of BRCA1 in complex human serum, indicating great potential of this novel biomaterial for application in biosensors and bioelectronics.

Epstein-Barr virus DNA loads in adult human immunodeficiency virus type 1-infected patients receiving highly active antiretroviral therapy

NASA Technical Reports Server (NTRS)

Ling, Paul D.; Vilchez, Regis A.; Keitel, Wendy A.; Poston, David G.; Peng, Rong Sheng; White, Zoe S.; Visnegarwala, Fehmida; Lewis, Dorothy E.; Butel, Janet S.

2003-01-01

Patients with human immunodeficiency virus type 1 (HIV-1) infection are at high risk of developing Epstein-Barr virus (EBV)-associated lymphoma. However, little is known of the EBV DNA loads in patients receiving highly active antiretroviral therapy (HAART). Using a real-time quantitative polymerase chain reaction assay, we demonstrated that significantly more HIV-1-infected patients receiving HAART than HIV-1-uninfected volunteers had detectable EBV DNA in blood (57 [81%] of 70 vs. 11 [16%] of 68 patients; P=.001) and saliva (55 [79%] of 68 vs. 37 [54%] of 68 patients; P=.002). The mean EBV loads in blood and saliva samples were also higher in HIV-1-infected patients than in HIV-1-uninfected volunteers (P=.001). The frequency of EBV detection in blood was associated with lower CD4+ cell counts (P=.03) among HIV-1-infected individuals, although no differences were observed in the EBV DNA loads in blood or saliva samples in the HIV-1-infected group. Additional studies are needed to determine whether EBV-specific CD4+ and CD8+ cells play a role in the pathogenesis of EBV in HIV-1-infected patients receiving HAART.

DNA methylation alterations in response to pesticide exposure in vitro

PubMed Central

Zhang, Xiao; Wallace, Andrew D.; Du, Pan; Kibbe, Warren A.; Jafari, Nadereh; Xie, Hehuang; Lin, Simon; Baccarelli, Andrea; Soares, Marcelo Bento; Hou, Lifang

2013-01-01

Although pesticides are subject to extensive carcinogenicity testing before regulatory approval, pesticide exposure has repeatedly been associated with various cancers. This suggests that pesticides may cause cancer via non-mutagenicity mechanisms. The present study provides evidence to support the hypothesis that pesticide-induced cancer may be mediated in part by epigenetic mechanisms. We examined whether exposure to 7 commonly used pesticides (i.e., fonofos, parathion, terbufos, chlorpyrifos, diazinon, malathion, and phorate) induces DNA methylation alterations in vitro. We conducted genome-wide DNA methylation analyses on DNA samples obtained from the human hematopoietic K562 cell line exposed to ethanol (control) and several OPs using the Illumina Infinium HumanMethylation27 BeadChip. Bayesian-adjusted t-tests were used to identify differentially methylated gene promoter CpG sites. In this report, we present our results on three pesticides (fonofos, parathion, and terbufos) that clustered together based on principle component analysis and hierarchical clustering. These three pesticides induced similar methylation changes in the promoter regions of 712 genes, while also exhibiting their own OP-specific methylation alterations. Functional analysis of methylation changes specific to each OP, or common to all three OPs, revealed that differential methylation was associated with numerous genes that are involved in carcinogenesis-related processes. Our results provide experimental evidence that pesticides may modify gene promoter DNA methylation levels, suggesting that epigenetic mechanisms may contribute to pesticide-induced carcinogenesis. Further studies in other cell types and human samples are required, as well as determining the impact of these methylation changes on gene expression. PMID:22847954

Using ancient DNA to study the origins and dispersal of ancestral Polynesian chickens across the Pacific

PubMed Central

Thomson, Vicki A.; Lebrasseur, Ophélie; Austin, Jeremy J.; Hunt, Terry L.; Burney, David A.; Denham, Tim; Rawlence, Nicolas J.; Wood, Jamie R.; Gongora, Jaime; Girdland Flink, Linus; Linderholm, Anna; Dobney, Keith; Larson, Greger; Cooper, Alan

2014-01-01

The human colonization of Remote Oceania remains one of the great feats of exploration in history, proceeding east from Asia across the vast expanse of the Pacific Ocean. Human commensal and domesticated species were widely transported as part of this diaspora, possibly as far as South America. We sequenced mitochondrial control region DNA from 122 modern and 22 ancient chicken specimens from Polynesia and Island Southeast Asia and used these together with Bayesian modeling methods to examine the human dispersal of chickens across this area. We show that specific techniques are essential to remove contaminating modern DNA from experiments, which appear to have impacted previous studies of Pacific chickens. In contrast to previous reports, we find that all ancient specimens and a high proportion of the modern chickens possess a group of unique, closely related haplotypes found only in the Pacific. This group of haplotypes appears to represent the authentic founding mitochondrial DNA chicken lineages transported across the Pacific, and allows the early dispersal of chickens across Micronesia and Polynesia to be modeled. Importantly, chickens carrying this genetic signature persist on several Pacific islands at high frequencies, suggesting that the original Polynesian chicken lineages may still survive. No early South American chicken samples have been detected with the diagnostic Polynesian mtDNA haplotypes, arguing against reports that chickens provide evidence of Polynesian contact with pre-European South America. Two modern specimens from the Philippines carry haplotypes similar to the ancient Pacific samples, providing clues about a potential homeland for the Polynesian chicken. PMID:24639505

TECHNICAL BRIEF: Isolation of total DNA from postmortem human eye tissues and quality comparison between iris and retina

PubMed Central

Wang, Jay Ching Chieh; Wang, Aikun; Gao, Jiangyuan; Cao, Sijia; Samad, Idris; Zhang, Dean; Ritland, Carol; Cui, Jing Z.

2012-01-01

Background Recent genomic technologies have propelled our understanding of the mechanisms underlying complex eye diseases such as age-related macular degeneration (AMD). Genotyping postmortem eye tissues for known single nucleotide polymorphisms (SNPs) associated with AMD may prove valuable, especially when combined with information obtained through other methods such as immunohistochemistry, western blot, enzyme-linked immunosorbent assay (ELISA), and proteomics. Initially intending to genotype postmortem eye tissues for AMD-related SNPs, our group became interested in isolating and comparing the quality of DNA from the iris and retina of postmortem donor eyes. Since there is no previously published protocol in the literature on this topic, we present a protocol suitable for isolating high-quality DNA from postmortem eye tissues for genomic studies. Methods DNA from 33 retinal samples and 35 iris samples was extracted using the phenol-chloroform-isoamyl method from postmortem donor eye tissues. The quantity of DNA was measured with a spectrophotometer while the quality was checked using gel electrophoresis. The DNA samples were then amplified with PCR for the complement factor H (CFH) gene. The purified amplified products were then genotyped for the SNPs in the CFH gene. Results Regarding concentration, the retina yielded 936 ng/μl of DNA, while the iris yielded 78 ng/μl of DNA. Retinal DNA was also purer than iris DNA (260/280=1.78 vs. 1.46, respectively), and produced superior PCR results. Retinal tissue yielded significantly more DNA than the iris tissue per mg of sample (21.7 ng/μl/mg vs. 7.42 ng/μl/mg). Retinal DNA can be readily amplified with PCR, while iris DNA can also be amplified by adding bovine serum albumin. Overall, retinal tissues yielded DNA of superior quality, quantity, and suitability for genotyping and genomic studies. Conclusions The protocol presented here provides a clear and reliable method for isolating total DNA from postmortem eye tissues. Retinal tissue provides DNA of excellent quantity and quality for genotyping and downstream genomic studies. However, DNA isolated from iris tissues, and treated with bovine serum albumin, may also be a valuable source of DNA for genotyping and genomic studies. PMID:23288996

75 FR 32191 - National Health and Nutrition Examination Survey (NHANES) DNA Samples: Guidelines for Proposals...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-07

... DEPARTMENT OF HEALTH AND HUMAN SERVICES Centers for Disease Control and Prevention National Health... Cost Schedule AGENCY: Centers for Disease Control and Prevention, Department of Health and Human... for Disease Control and Prevention (CDC). Examination surveys conducted since 1960 by NCHS have...

Low prevalence of transcriptionally active human papilloma virus in Indian patients with HNSCC and leukoplakia.

PubMed

Bhosale, Priyanka G; Pandey, Manishkumar; Desai, Rajiv S; Patil, Asawari; Kane, Shubhada; Prabhash, Kumar; Mahimkar, Manoj B

2016-11-01

In the present study, we comprehensively analyzed the prevalence of transcriptionally active human papilloma virus (HPV) in tissue samples of Indian patients with leukoplakia, predominantly hyperplastic lesions and head and neck squamous cell carcinoma (HNSCC). In addition, saliva samples from patients with HNSCC were screened for HPV detection. P16 overexpression was analyzed by immunohistochemistry. Tissue samples of leukoplakia (n = 121) and HNSCC (n = 427) and saliva from patients with HNSCC (n = 215) were tested for HPV using nested polymerase chain reaction. Positive samples were sequenced for subtyping. The presence of HPV E6/E7 mRNA was confirmed by RNA in situ hybridization. P16 expression and HPV DNA were not detected in any of the leukoplakia specimens. Of the 427 HNSCC tumors, 9 showed p16 overexpression and 7/427 cases were positive for HPV16 DNA, in saliva or tissue. E6/E7 mRNA positivity was observed in 8 HNSCC samples, primarily from patients with no habit of tobacco consumption. The prevalence of high-risk HPV was restricted to oropharynx and larynx, with very little concordance between p16 overexpression and HPV positivity. All patients with HPV-positive saliva samples had transcriptionally active HPV present in their tumors. The presence of HPV DNA does not necessarily reflect transcriptionally active virus in tumors; hence, it is important to consider this fact while categorizing HPV-associated tumors. Copyright © 2016 Elsevier Inc. All rights reserved.

Performance of the ForenSeqTM DNA Signature Prep kit on highly degraded samples.

PubMed

Fattorini, Paolo; Previderé, Carlo; Carboni, Ilaria; Marrubini, Giorgio; Sorçaburu-Cigliero, Solange; Grignani, Pierangela; Bertoglio, Barbara; Vatta, Paolo; Ricci, Ugo

2017-04-01

Next generation sequencing (NGS) is the emerging technology in forensic genomics laboratories. It offers higher resolution to address most problems of human identification, greater efficiency and potential ability to interrogate very challenging forensic casework samples. In this study, a trial set of DNA samples was artificially degraded by progressive aqueous hydrolysis, and analyzed together with the corresponding unmodified DNA sample and control sample 2800 M, to test the performance and reliability of the ForenSeq TM DNA Signature Prep kit using the MiSeq Sequencer (Illumina). The results of replicate tests performed on the unmodified sample (1.0 ng) and on scalar dilutions (1.0, 0.5 and 0.1 ng) of the reference sample 2800 M showed the robustness and the reliability of the NGS approach even from sub-optimal amounts of high quality DNA. The degraded samples showed a very limited number of reads/sample, from 2.9-10.2 folds lower than the ones reported for the less concentrated 2800 M DNA dilution (0.1 ng). In addition, it was impossible to assign up to 78.2% of the genotypes in the degraded samples as the software identified the corresponding loci as "low coverage" (< 50x). Amplification artifacts such as allelic imbalances, allele drop outs and a single allele drop in were also scored in the degraded samples. However, the ForenSeq TM DNA Sequencing kit, on the Illumina MiSeq, was able to generate data which led to the correct typing of 5.1-44.8% and 10.9-58.7% of 58 of the STRs and 92 SNPs, respectively. In all trial samples, the SNP markers showed higher chances to be typed correctly compared to the STRs. This NGS approach showed very promising results in terms of ability to recover genetic information from heavily degraded DNA samples for which the conventional PCR/CE approach gave no results. The frequency of genetic mistyping was very low, reaching the value of 1.4% for only one of the degraded samples. However, these results suggest that further validation studies and a definition of interpretation criteria for NGS data are needed before implementation of this technique in forensic genetics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of the DNA damaging potential of cannabis cigarette smoke by the determination of acetaldehyde derived N2-ethyl-2'-deoxyguanosine adducts.

PubMed

Singh, Rajinder; Sandhu, Jatinderpal; Kaur, Balvinder; Juren, Tina; Steward, William P; Segerbäck, Dan; Farmer, Peter B

2009-06-01

Acetaldehyde is an ubiquitous genotoxic compound that has been classified as a possible carcinogen to humans. It can react with DNA to form primarily a Schiff base N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG) adduct. An online column-switching valve liquid chromatography tandem mass spectrometry (LC-MS/MS) selected reaction monitoring (SRM) method was developed for the determination of N(2)-ethylidene-dG adducts in DNA following reduction with sodium cyanoborohydride (NaBH(3)CN) to the chemically stable N(2)-ethyl-2'-deoxyguanosine (N(2)-ethyl-dG) adduct. Accurate quantitation of the adduct was obtained by the addition of the [(15)N(5)]N(2)-ethyl-dG stable isotope-labeled internal standard prior to enzymatic hydrolysis of the DNA samples to 2'-deoxynucleosides with the incorporation of NaBH(3)CN in the DNA hydrolysis buffer. The method required 50 microg of hydrolyzed DNA on column for the analysis, and the limit of detection for N(2)-ethyl-dG was 2.0 fmol. The analysis of calf thymus DNA treated in vitro with acetaldehyde (ranging from 0.5 to 100 mM) or with the smoke generated from 1, 5, and 10 cannabis cigarettes showed linear dose-dependent increases in the level of N(2)-ethyl-dG adducts (r = 0.954 and r = 0.999, respectively). Similar levels (332.8 +/- 21.9 vs 348.4 +/- 19.1 adducts per 10(8) 2'-deoxynucleosides) of N(2)-ethyl-dG adducts were detected following the exposure of calf thymus DNA to 10 tobacco or 10 cannabis cigarettes. No significant difference was found in the levels of N(2)-ethyl-dG adducts in human lung DNA obtained from nonsmokers (n = 4) and smokers (n = 4) with the average level observed as 13.3 +/- 0.7 adducts per 10(8) 2'-deoxynucleosides. No N(2)-ethyl-dG adducts were detected in any of the DNA samples following analysis with the omission of NaBH(3)CN from the DNA hydrolysis buffer. In conclusion, these results provide evidence for the DNA damaging potential of cannabis smoke, implying that the consumption of cannabis cigarettes may be detrimental to human health with the possibility to initiate cancer development.

Chicken skin virome analyzed by high-throughput sequencing shows a composition highly different from human skin.

PubMed

Denesvre, Caroline; Dumarest, Marine; Rémy, Sylvie; Gourichon, David; Eloit, Marc

2015-10-01

Recent studies show that human skin at homeostasis is a complex ecosystem whose virome include circular DNA viruses, especially papillomaviruses and polyomaviruses. To determine the chicken skin virome in comparison with human skin virome, a chicken swabs pool sample from fifteen indoor healthy chickens of five genetic backgrounds was examined for the presence of DNA viruses by high-throughput sequencing (HTS). The results indicate a predominance of herpesviruses from the Mardivirus genus, coming from either vaccinal origin or presumably asymptomatic infection. Despite the high sensitivity of the HTS method used herein to detect small circular DNA viruses, we did not detect any papillomaviruses, polyomaviruses, or circoviruses, indicating that these viruses may not be resident of the chicken skin. The results suggest that the turkey herpesvirus is a resident of chicken skin in vaccinated chickens. This study indicates major differences between the skin viromes of chickens and humans. The origin of this difference remains to be further studied in relation with skin physiology, environment, or virus population dynamics.

Detection of human Pneumocystis carinii by the polymerase chain reaction.

PubMed

Becker-Hapak, M; Liberator, P; Graves, D

1991-01-01

Oligonucleotide primers were prepared from a clone (B12) which has been shown to be a repetitive sequence in the rat P. carinii genome. Polymerase chain reaction was employed to amplify both rat and human P. carinii DNA. The detection limit of the assay was approximately 600 ng of total nucleic acid. Amplification products from both the rat and human isolates (ca. 780 bp) were characterized by denaturing gradient gel electrophoresis after digestion with Sau3A. No amplification products were obtained when DNA from the following potential pulmonary pathogens were used in identical reactions: Aspergillus fumigatus, Cryptococcus neoformans, Candida albicans, Mycobacterium avium-intracellulare and cytomegalovirus. In a blind study using the B12 primers, P. carinii DNA was successfully amplified in clinical samples which were positive by direct immunofluorescence assay (IFA) as well as in some specimens not identified by direct IFA.

DNA detection rates of host mtDNA in bloodmeals of human body lice (Pediculus humanus L., 1758).

PubMed

Davey, J S; Casey, C S; Burgess, I F; Cable, J

2007-09-01

Using polymerase chain reaction, we investigated the extent to which digestion affects the potential to amplify 12S mitochondrial DNA sequences from bloodmeals of individual human body lice (Pediculus humanus L.) (Phthiraptera, Pediculidae) up to 72 h after feeding on a surrogate rabbit host (Oryctolagus cuniculus L.) (Lagomorpha, Leporidae). Two rabbit-specific primer pairs were developed to produce amplicons of 199 bp and 283 bp, the smaller of which was found to have a significantly slower decay rate. Median detection periods (T50) for the amplicons were 20 h and 12 h, with maximum detection periods of 24 h and 12 h, respectively, suggesting an inversely proportional linear relationship between amplicon size and digestion time. The data provide an indication of timeframes essential for the design of forensic sampling protocols and a basis for investigating the feeding frequency of human lice.

Optimal Ancient DNA Yields from the Inner Ear Part of the Human Petrous Bone.

PubMed

Pinhasi, Ron; Fernandes, Daniel; Sirak, Kendra; Novak, Mario; Connell, Sarah; Alpaslan-Roodenberg, Songül; Gerritsen, Fokke; Moiseyev, Vyacheslav; Gromov, Andrey; Raczky, Pál; Anders, Alexandra; Pietrusewsky, Michael; Rollefson, Gary; Jovanovic, Marija; Trinhhoang, Hiep; Bar-Oz, Guy; Oxenham, Marc; Matsumura, Hirofumi; Hofreiter, Michael

2015-01-01

The invention and development of next or second generation sequencing methods has resulted in a dramatic transformation of ancient DNA research and allowed shotgun sequencing of entire genomes from fossil specimens. However, although there are exceptions, most fossil specimens contain only low (~ 1% or less) percentages of endogenous DNA. The only skeletal element for which a systematically higher endogenous DNA content compared to other skeletal elements has been shown is the petrous part of the temporal bone. In this study we investigate whether (a) different parts of the petrous bone of archaeological human specimens give different percentages of endogenous DNA yields, (b) there are significant differences in average DNA read lengths, damage patterns and total DNA concentration, and (c) it is possible to obtain endogenous ancient DNA from petrous bones from hot environments. We carried out intra-petrous comparisons for ten petrous bones from specimens from Holocene archaeological contexts across Eurasia dated between 10,000-1,800 calibrated years before present (cal. BP). We obtained shotgun DNA sequences from three distinct areas within the petrous: a spongy part of trabecular bone (part A), the dense part of cortical bone encircling the osseous inner ear, or otic capsule (part B), and the dense part within the otic capsule (part C). Our results confirm that dense bone parts of the petrous bone can provide high endogenous aDNA yields and indicate that endogenous DNA fractions for part C can exceed those obtained for part B by up to 65-fold and those from part A by up to 177-fold, while total endogenous DNA concentrations are up to 126-fold and 109-fold higher for these comparisons. Our results also show that while endogenous yields from part C were lower than 1% for samples from hot (both arid and humid) parts, the DNA damage patterns indicate that at least some of the reads originate from ancient DNA molecules, potentially enabling ancient DNA analyses of samples from hot regions that are otherwise not amenable to ancient DNA analyses.

Application of a loop-mediated isothermal amplification (LAMP) assay targeting cox1 gene for the detection of Clonorchis sinensis in human fecal samples

PubMed Central

Rahman, S. M. Mazidur; Song, Hyun Beom; Jin, Yan; Oh, Jin-Kyoung; Lim, Min Kyung; Hong, Sung-Tae

2017-01-01

Background Clonorchiasis is prevalent in the Far East, and a major health problem in endemic areas. Infected persons may experience, if not treated, serious complications such as bile stone formation, pyogenic cholangitis, and even cholangiocarcinoma. Early diagnosis and treatment are important to prevent serious complications and, therefore, the simple and reliable diagnostic method is necessary to control clonorchiasis in endemic areas, where resources for the diagnosis are limited. Methodology/Principle findings The loop-mediated isothermal amplification (LAMP) assay has been applied for the detection of Clonorchis sinensis DNA. Six primers targeting eight locations on the cytochrome c oxidase subunit 1 gene of C. sinensis were designed for species-specific amplification using the LAMP assay. The LAMP assay was sensitive enough to detect as little as 100 fg of C. sinensis genomic DNA and the detection limit in 100 mg of stool was as low as one egg. The assay was highly specific because no cross-reactivity was observed with the DNA of other helminths, protozoa or Escherichia coli. Then, LAMP assay was applied to human fecal samples collected from an endemic area of clonorchiasis in Korea. Using samples showing consistent results by both Kato-Katz method and real-time PCR as reference standards, the LAMP assay showed 97.1% (95% CI, 90.1–99.2) of sensitivity and 100% (95% CI, 92.9–100) of specificity. In stool samples with more than 100 eggs per gram of feces, the sensitivity achieved 100%. Conclusions To detect C. sinensis in human fecal samples, the LAMP assay was applied and achieved high sensitivity and specificity. The LAMP assay can be utilized in field laboratories as a powerful tool for diagnosis and epidemiological survey of clonorchiasis. PMID:28991924

Application of a loop-mediated isothermal amplification (LAMP) assay targeting cox1 gene for the detection of Clonorchis sinensis in human fecal samples.

PubMed

Rahman, S M Mazidur; Song, Hyun Beom; Jin, Yan; Oh, Jin-Kyoung; Lim, Min Kyung; Hong, Sung-Tae; Choi, Min-Ho

2017-10-01

Clonorchiasis is prevalent in the Far East, and a major health problem in endemic areas. Infected persons may experience, if not treated, serious complications such as bile stone formation, pyogenic cholangitis, and even cholangiocarcinoma. Early diagnosis and treatment are important to prevent serious complications and, therefore, the simple and reliable diagnostic method is necessary to control clonorchiasis in endemic areas, where resources for the diagnosis are limited. The loop-mediated isothermal amplification (LAMP) assay has been applied for the detection of Clonorchis sinensis DNA. Six primers targeting eight locations on the cytochrome c oxidase subunit 1 gene of C. sinensis were designed for species-specific amplification using the LAMP assay. The LAMP assay was sensitive enough to detect as little as 100 fg of C. sinensis genomic DNA and the detection limit in 100 mg of stool was as low as one egg. The assay was highly specific because no cross-reactivity was observed with the DNA of other helminths, protozoa or Escherichia coli. Then, LAMP assay was applied to human fecal samples collected from an endemic area of clonorchiasis in Korea. Using samples showing consistent results by both Kato-Katz method and real-time PCR as reference standards, the LAMP assay showed 97.1% (95% CI, 90.1-99.2) of sensitivity and 100% (95% CI, 92.9-100) of specificity. In stool samples with more than 100 eggs per gram of feces, the sensitivity achieved 100%. To detect C. sinensis in human fecal samples, the LAMP assay was applied and achieved high sensitivity and specificity. The LAMP assay can be utilized in field laboratories as a powerful tool for diagnosis and epidemiological survey of clonorchiasis.

Levels of select PCB and PBDE congeners in human post-mortem brain reveal possible environmental involvement in 15q11-q13 duplication autism spectrum disorder

PubMed Central

Mitchell, Michelle M.; Woods, Rima; Chi, Lai-Har; Schmidt, Rebecca J.; Pessah, Isaac N.; Kostyniak, Paul J.; LaSalle, Janine M.

2013-01-01

Persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs) that bioaccumulate in lipid-rich tissues are of concern as developmental neurotoxicants. Epigenetic mechanisms such as DNA methylation act at the interface of genetic and environmental factors implicated in autism-spectrum disorders. The relationship between POP levels and DNA methylation patterns in individuals with and without neurodevelopmental disorders has not been previously investigated. In this study, a total of 107 human frozen post-mortem brain samples were analyzed for 8 PCBs and 7 PBDEs by GC-micro electron capture detector and GC/MS using negative chemical ionization. Human brain samples were grouped as neurotypical controls (n=43), neurodevelopmental disorders with known genetic basis (n=32, including Down, Rett, Prader-Willi, Angelman, and 15q11-q13 duplication syndromes), and autism of unknown etiology (n=32). Unexpectedly, PCB 95 was significantly higher in the genetic neurodevelopmental group, but not idiopathic autism, as compared to neurotypical controls. Interestingly, samples with detectable PCB 95 levels were almost exclusively those with maternal 15q11-q13 duplication (Dup15q) or deletion in Prader-Willi syndrome. When sorted by birth year, Dup15q samples represented five out of six of genetic neurodevelopmental samples born after the 1976 PCB ban exhibiting detectable PCB 95 levels. Dup15q was the strongest predictor of PCB 95 exposure over age, gender, or year of birth. Dup15q brain showed lower levels of repetitive DNA methylation measured by LINE-1 pyrosequencing, but methylation levels were confounded by year of birth. These results demonstrate a novel paradigm by which specific POPs may predispose to genetic copy number variation of 15q11-q13. PMID:22930557

Lactobacilli and Bifidobacteria in Human Breast Milk: Influence of Antibiotherapy and Other Host and Clinical Factors

PubMed Central

Soto, Ana; Martín, Virginia; Jiménez, Esther; Mader, Isabelle; Rodríguez, Juan M.; Fernández, Leonides

2014-01-01

ABSTRACT Objective: The objective of this work was to study the lactobacilli and bifidobacteria population in human milk of healthy women, and to investigate the influence that several factors (including antibioteraphy during pregnancy and lactation, country and date of birth, delivery mode, or infant age) may exert on such population. Methods: A total of 160 women living in Germany or Austria provided the breast milk samples. Initially, 66 samples were randomly selected and cultured on MRS-Cys agar plates. Then, the presence of DNA from the genera Lactobacillus and Bifidobacterium, and from most of the Lactobacillus and Bifidobacterium species that were isolated, was assessed by qualitative polymerase chain reaction (PCR) using genus- and species-specific primers. Results: Lactobacilli and bifidobacteria could be isolated from the milk of 27 (40.91%) and 7 (10.61%), respectively, of the 66 cultured samples. On the contrary, Lactobacillus and Bifidobacterium sequences were detected by PCR in 108 (67.50%) and 41 (25.62%), respectively, of the 160 samples analyzed. The Lactobacillus species most frequently isolated and detected was L salivarius (35.00%), followed by L fermentum (25.00%) and L gasseri (21.88%), whereas B breve (13.75%) was the bifidobacterial species most commonly recovered and whose DNA was most regularly found. The number of lactobacilli- or bifidobacteria-positive samples was significantly lower in women who had received antibiotherapy during pregnancy or lactation. Conclusions: Our results suggest that either the presence of lactobacilli and/or bifidobacteria or their DNA may constitute good markers of a healthy human milk microbiota that has not been altered by the use of antibiotics. PMID:24590211

Developmental validation of the MiSeq FGx Forensic Genomics System for Targeted Next Generation Sequencing in Forensic DNA Casework and Database Laboratories.

PubMed

Jäger, Anne C; Alvarez, Michelle L; Davis, Carey P; Guzmán, Ernesto; Han, Yonmee; Way, Lisa; Walichiewicz, Paulina; Silva, David; Pham, Nguyen; Caves, Glorianna; Bruand, Jocelyne; Schlesinger, Felix; Pond, Stephanie J K; Varlaro, Joe; Stephens, Kathryn M; Holt, Cydne L

2017-05-01

Human DNA profiling using PCR at polymorphic short tandem repeat (STR) loci followed by capillary electrophoresis (CE) size separation and length-based allele typing has been the standard in the forensic community for over 20 years. Over the last decade, Next-Generation Sequencing (NGS) matured rapidly, bringing modern advantages to forensic DNA analysis. The MiSeq FGx™ Forensic Genomics System, comprised of the ForenSeq™ DNA Signature Prep Kit, MiSeq FGx™ Reagent Kit, MiSeq FGx™ instrument and ForenSeq™ Universal Analysis Software, uses PCR to simultaneously amplify up to 231 forensic loci in a single multiplex reaction. Targeted loci include Amelogenin, 27 common, forensic autosomal STRs, 24 Y-STRs, 7 X-STRs and three classes of single nucleotide polymorphisms (SNPs). The ForenSeq™ kit includes two primer sets: Amelogenin, 58 STRs and 94 identity informative SNPs (iiSNPs) are amplified using DNA Primer Set A (DPMA; 153 loci); if a laboratory chooses to generate investigative leads using DNA Primer Set B, amplification is targeted to the 153 loci in DPMA plus 22 phenotypic informative (piSNPs) and 56 biogeographical ancestry SNPs (aiSNPs). High-resolution genotypes, including detection of intra-STR sequence variants, are semi-automatically generated with the ForenSeq™ software. This system was subjected to developmental validation studies according to the 2012 Revised SWGDAM Validation Guidelines. A two-step PCR first amplifies the target forensic STR and SNP loci (PCR1); unique, sample-specific indexed adapters or "barcodes" are attached in PCR2. Approximately 1736 ForenSeq™ reactions were analyzed. Studies include DNA substrate testing (cotton swabs, FTA cards, filter paper), species studies from a range of nonhuman organisms, DNA input sensitivity studies from 1ng down to 7.8pg, two-person human DNA mixture testing with three genotype combinations, stability analysis of partially degraded DNA, and effects of five commonly encountered PCR inhibitors. Calculations from ForenSeq™ STR and SNP repeatability and reproducibility studies (1ng template) indicate 100.0% accuracy of the MiSeq FGx™ System in allele calling relative to CE for STRs (1260 samples), and >99.1% accuracy relative to bead array typing for SNPs (1260 samples for iiSNPs, 310 samples for aiSNPs and piSNPs), with >99.0% and >97.8% precision, respectively. Call rates of >99.0% were observed for all STRs and SNPs amplified with both ForenSeq™ primer mixes. Limitations of the MiSeq FGx™ System are discussed. Results described here demonstrate that the MiSeq FGx™ System meets forensic DNA quality assurance guidelines with robust, reliable, and reproducible performance on samples of various quantities and qualities. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Detection of human papillomavirus (HPV) DNA in human prostatic tissues by polymerase chain reaction (PCR).

PubMed

Sarkar, F H; Sakr, W A; Li, Y W; Sreepathi, P; Crissman, J D

1993-01-01

Human papillomavirus (HPV) infections are strongly linked to the pathogenesis of uterine cervical neoplasms, and have been implicated in other cancers of the female genital tract. In contrast, the association of HPV with the cancers of the male urogenital tract is less evident, except in anal and penile cancers. However, recent studies reporting the prevalence of HPV infections in human prostate cancers (60-100% HPV 16 positive vs. no infection of HPV) have raised controversies regarding the prevalence of HPV in benign and neoplastic human prostate. We investigated the prevalence of HPV infections in prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinomas in 23 surgically resected prostates. Polymerase chain reaction (PCR) was used to amplify HPV 6b/11, 16, and 18 specific DNA sequences, using type specific HPV primers selected from the transforming gene E6-E7. The areas of PIN and cancer in 6 microns H&E stained tissue sections were identified, and respective areas of PIN and cancer were isolated from the adjacent serial sections and used for DNA amplification and HPV detection (Fig. 1). Our results demonstrated the presence of HPV 16 in three carcinomas (13%), using type specific primers in PCR amplified samples. We were not able to demonstrate the presence of other HPV types (HPV 6b/11 or HPV 18) in any of the samples using specific primers. Two of these prostates showed relatively strong positive signals by dot blot analysis, when hybridized with a 32P-labeled HPV 16 type specific oligonucleotide probe. One more sample showed weak positivity, when hybridized with a 32P-labeled HPV 16 type specific oligonucleotide probe. Subsequently, we have confirmed these results by Southern hybridization of the samples transferred to nylon membrane after agarose gel electrophoresis and detected by HPV 16 type specific oligonucleotide probe, using chemiluminescent assay. We, therefore, conclude that HPV infections of the prostate in general are not as common as has been previously claimed by other investigators.

Whole genome nucleosome sequencing identifies novel types of forensic markers in degraded DNA samples

PubMed Central

Dong, Chun-nan; Yang, Ya-dong; Li, Shu-jin; Yang, Ya-ran; Zhang, Xiao-jing; Fang, Xiang-dong; Yan, Jiang-wei; Cong, Bin

2016-01-01

In the case of mass disasters, missing persons and forensic caseworks, highly degraded biological samples are often encountered. It can be a challenge to analyze and interpret the DNA profiles from these samples. Here we provide a new strategy to solve the problem by taking advantage of the intrinsic structural properties of DNA. We have assessed the in vivo positions of more than 35 million putative nucleosome cores in human leukocytes using high-throughput whole genome sequencing, and identified 2,462 single nucleotide variations (SNVs), 128 insertion-deletion polymorphisms (indels). After comparing the sequence reads with 44 STR loci commonly used in forensics, five STRs (TH01, TPOX, D18S51, DYS391, and D10S1248)were matched. We compared these “nucleosome protected STRs” (NPSTRs) with five other non-NPSTRs using mini-STR primer design, real-time PCR, and capillary gel electrophoresis on artificially degraded DNA. Moreover, genotyping performance of the five NPSTRs and five non-NPSTRs was also tested with real casework samples. All results show that loci located in nucleosomes are more likely to be successfully genotyped in degraded samples. In conclusion, after further strict validation, these markers could be incorporated into future forensic and paleontology identification kits, resulting in higher discriminatory power for certain degraded sample types. PMID:27189082

The performance of multimodal hyperspectral spectroscopy in the detection of precancerous cervical lesions

NASA Astrophysics Data System (ADS)

Trahmono; Lusiana, N.; Indarti, J.

2017-08-01

The aim of this study was to compare the performance of multimodal hyperspectral spectroscopy (MHS), which combines fluorescence and reflectance spectroscopy, with that of conventional laboratory-based screening tests, such as the Papanicolaou (Pap) smear test and human papilloma virus (HPV) DNA test, for detecting precancerous lesions of the cervix. The study utilized a cross-sectional design, and the kappa test was used in the analytical assessment. MHS scans were obtained from a sample of 70 consecutive patients, followed by sample collection for Pap and HPV DNA analysis and colposcopy referral, if indicated. Of the 70 patients evaluated, the results of cervical spectroscopy were normal in 38 (54.3%) patients, and they were abnormal in 32 (45.7%) patients. Based on the cytology results, 45 (64.3%) samples were normal, and 25 (35.7%) samples were abnormal. According to the results of the HPV DNA test, 47 (67.14%) samples were normal, and 17 (24.28%) samples were abnormal. Based on the results of the kappa test, the agreement between MHS and cytology was 0.793 (p < 0.001). The agreement between MHS and the HPV DNA test was 0.195 (p = 0.086), and the agreement between MHS and colposcopy was 0.479 (p < 0.001).

Direct and long-term detection of gene doping in conventional blood samples.

PubMed

Beiter, T; Zimmermann, M; Fragasso, A; Hudemann, J; Niess, A M; Bitzer, M; Lauer, U M; Simon, P

2011-03-01

The misuse of somatic gene therapy for the purpose of enhancing athletic performance is perceived as a coming threat to the world of sports and categorized as 'gene doping'. This article describes a direct detection approach for gene doping that gives a clear yes-or-no answer based on the presence or absence of transgenic DNA in peripheral blood samples. By exploiting a priming strategy to specifically amplify intronless DNA sequences, we developed PCR protocols allowing the detection of very small amounts of transgenic DNA in genomic DNA samples to screen for six prime candidate genes. Our detection strategy was verified in a mouse model, giving positive signals from minute amounts (20 μl) of blood samples for up to 56 days following intramuscular adeno-associated virus-mediated gene transfer, one of the most likely candidate vector systems to be misused for gene doping. To make our detection strategy amenable for routine testing, we implemented a robust sample preparation and processing protocol that allows cost-efficient analysis of small human blood volumes (200 μl) with high specificity and reproducibility. The practicability and reliability of our detection strategy was validated by a screening approach including 327 blood samples taken from professional and recreational athletes under field conditions.

Palaeoproteomics for human evolution studies

NASA Astrophysics Data System (ADS)

Welker, Frido

2018-06-01

The commonplace sequencing of Neanderthal, Denisovan and ancient modern human DNA continues to revolutionize our understanding of hominin phylogeny and interaction(s). The challenge with older fossils is that the progressive fragmentation of DNA even under optimal conditions, a function of time and temperature, results in ever shorter fragments of DNA. This process continues until no DNA can be sequenced or reliably aligned. Ancient proteins ultimately suffer a similar fate, but are a potential alternative source of biomolecular sequence data to investigate hominin phylogeny given their slower rate of fragmentation. In addition, ancient proteins have been proposed to potentially provide insights into in vivo biological processes and can be used to provide additional ecological information through large scale ZooMS (Zooarchaeology by Mass Spectrometry) screening of unidentifiable bone fragments. However, as initially with ancient DNA, most ancient protein research has focused on Late Pleistocene or Holocene samples from Europe. In addition, only a limited number of studies on hominin remains have been published. Here, an updated review on ancient protein analysis in human evolutionary contexts is given, including the identification of specific knowledge gaps and existing analytical limits, as well as potential avenues to overcome these.

Evaluating differential nuclear DNA yield rates and osteocyte numbers among human bone tissue types: A synchrotron radiation micro-CT approach.

PubMed

Andronowski, Janna M; Mundorff, Amy Z; Pratt, Isaac V; Davoren, Jon M; Cooper, David M L

2017-05-01

Molecular human identification has conventionally focused on DNA sampling from dense, weight-bearing cortical bone tissue, typically from femora or tibiae. A comparison of skeletal elements from three contemporary individuals demonstrated that elements with high quantities of cancellous bone yielded nuclear DNA at the highest rates, suggesting that preferentially sampling cortical bone may be suboptimal (Mundorff & Davoren, 2014). Despite these findings, the reason for the differential DNA yields between cortical and cancellous bone tissues remains unknown. The primary goal of this work is to ascertain whether differences in bone microstructure can be used to explain differential nuclear DNA yield among bone tissue types observed by Mundorff and Davoren (2014), with a focus on osteocytes and the three-dimensional (3D) quantification of their associated lacunae. Osteocytes and other bone cells are recognized to house DNA in bone tissue, thus examining the density of their lacunae may explain why nuclear DNA yield rates differ among bone tissue types. Lacunae were visualized and quantified using synchrotron radiation-based micro-Computed Tomographic imaging (SR micro-CT). Volumes of interest (VOIs) from cortical and cancellous bone tissues (n=129) were comparatively analyzed from the three skeletons sampled for Mundorff and Davoren's (2014) study. Analyses tested the primary hypothesis that the abundance and density of osteocytes (inferred from their lacunar spaces) vary between cortical and cancellous bone tissue types. Results demonstrated that osteocyte lacunar abundance and density vary between cortical and cancellous bone tissue types, with cortical bone VOIs containing a higher lacunar abundance and density. We found that the osteocyte lacunar density values are independent of nuclear DNA yield, suggesting an alternative explanation for the higher nuclear DNA yields from bones with greater quantities of cancellous bone tissue. The use of SR micro-CT allowed for a scale of analysis that revealed a high range of variation in lacunar abundance in both tissue types. Moreover, high-resolution SR micro-CT imaging revealed potential soft tissue remnants within marrow spaces not visible macroscopically. It is hypothesized that soft tissue remnants observed among the trabeculae of skeletal elements with high quantities of cancellous bone tissue are responsible for the high nuclear DNA yields. These findings have significant implications for bone-sample selection for nuclear DNA analysis in a forensic context when skeletal remains are recovered from the ground surface. Copyright © 2017 Elsevier B.V. All rights reserved.

Nonisotopic detection of human papillomavirus DNA in clinical specimens using a consensus PCR and a generic probe mix in an enzyme-linked immunosorbent assay format.

PubMed

Kornegay, J R; Shepard, A P; Hankins, C; Franco, E; Lapointe, N; Richardson, H; Coutleé, F

2001-10-01

We assessed the value of a new digoxigenin (DIG)-labeled generic probe mix in a PCR-enzyme-linked immunosorbent assay format to screen for the presence of human papillomavirus (HPV) DNA amplified from clinical specimens. After screening with this new generic assay is performed, HPV DNA-positive samples can be directly genotyped using a reverse blotting method with product from the same PCR amplification. DNA from 287 genital specimens was amplified via PCR using biotin-labeled consensus primers directed to the L1 gene. HPV amplicons were captured on a streptavidin-coated microwell plate (MWP) and detected with a DIG-labeled HPV generic probe mix consisting of nested L1 fragments from types 11, 16, 18, and 51. Coamplification and detection of human DNA with biotinylated beta-globin primers served as a control for both sample adequacy and PCR amplification. All specimens were genotyped using a reverse line blot assay (13). Results for the generic assay using MWPs and a DIG-labeled HPV generic probe mix (DIG-MWP generic probe assay) were compared with results from a previous analysis using dot blots with a radiolabeled nested generic probe mix and type-specific probes for genotyping. The DIG-MWP generic probe assay resulted in high intralaboratory concordance in genotyping results (88% versus 73% agreement using traditional methods). There were 207 HPV-positive results using the DIG-MWP method and 196 positives using the radiolabeled generic probe technique, suggesting slightly improved sensitivity. Only one sample failed to test positive with the DIG-MWP generic probe assay in spite of a positive genotyping result. Concordance between the two laboratories was nearly 87%. Approximately 6% of samples that were positive or borderline when tested with the DIG-MWP generic probe assay were not detected with the HPV type-specific panel, perhaps representing very rare or novel HPV types. This new method is easier to perform than traditional generic probe techniques and uses more objective interpretation criteria, making it useful in studies of HPV natural history.

Microfluidic Devices for Forensic DNA Analysis: A Review

PubMed Central

Bruijns, Brigitte; van Asten, Arian; Tiggelaar, Roald; Gardeniers, Han

2016-01-01

Microfluidic devices may offer various advantages for forensic DNA analysis, such as reduced risk of contamination, shorter analysis time and direct application at the crime scene. Microfluidic chip technology has already proven to be functional and effective within medical applications, such as for point-of-care use. In the forensic field, one may expect microfluidic technology to become particularly relevant for the analysis of biological traces containing human DNA. This would require a number of consecutive steps, including sample work up, DNA amplification and detection, as well as secure storage of the sample. This article provides an extensive overview of microfluidic devices for cell lysis, DNA extraction and purification, DNA amplification and detection and analysis techniques for DNA. Topics to be discussed are polymerase chain reaction (PCR) on-chip, digital PCR (dPCR), isothermal amplification on-chip, chip materials, integrated devices and commercially available techniques. A critical overview of the opportunities and challenges of the use of chips is discussed, and developments made in forensic DNA analysis over the past 10–20 years with microfluidic systems are described. Areas in which further research is needed are indicated in a future outlook. PMID:27527231

The implications of shedder status and background DNA on direct and secondary transfer in an attack scenario.

PubMed

Fonneløp, Ane Elida; Ramse, Merete; Egeland, Thore; Gill, Peter

2017-07-01

In court questions are often raised related to how trace DNA was deposited, directly during the crime or innocently for instance by secondary transfer. It is therefore of interest to have knowledge of the probability of transfer or secondary transfer in different situations. Factors that could influence transfer probabilities are background DNA and the shedder status of the involved persons. In this study, we have classified participants as high or low DNA shedders. We observed DNA transfer in a simulated attack scenario, and demonstrated that shedder status has a significant influence of transfer rates. We have examined the background DNA in samples from T-shirts worn in an area with frequent human traffic and detected multiple contributors. We further demonstrated that DNA from co-workers of a T-shirt wearer can be secondarily transferred from the environment and detected in samples, and that the composition of background DNA is correlated with the shedder status of the wearer. Finally, we have illustrated the inference with the results of transfer probabilities and a fictive case with the use of a Bayesian network. Copyright © 2017 Elsevier B.V. All rights reserved.

Mitochondrial sequence analysis for forensic identification using pyrosequencing technology.

PubMed

Andréasson, H; Asp, A; Alderborn, A; Gyllensten, U; Allen, M

2002-01-01

Over recent years, requests for mtDNA analysis in the field of forensic medicine have notably increased, and the results of such analyses have proved to be very useful in forensic cases where nuclear DNA analysis cannot be performed. Traditionally, mtDNA has been analyzed by DNA sequencing of the two hypervariable regions, HVI and HVII, in the D-loop. DNA sequence analysis using the conventional Sanger sequencing is very robust but time consuming and labor intensive. By contrast, mtDNA analysis based on the pyrosequencing technology provides fast and accurate results from the human mtDNA present in many types of evidence materials in forensic casework. The assay has been developed to determine polymorphic sites in the mitochondrial D-loop as well as the coding region to further increase the discrimination power of mtDNA analysis. The pyrosequencing technology for analysis of mtDNA polymorphisms has been tested with regard to sensitivity, reproducibility, and success rate when applied to control samples and actual casework materials. The results show that the method is very accurate and sensitive; the results are easily interpreted and provide a high success rate on casework samples. The panel of pyrosequencing reactions for the mtDNA polymorphisms were chosen to result in an optimal discrimination power in relation to the number of bases determined.

Aedes albopictus and Culex pipiens implicated as natural vectors of Dirofilaria repens in central Italy.

PubMed

Cancrini, G; Scaramozzino, P; Gabrielli, S; Di Paolo, M; Toma, L; Romi, R

2007-11-01

To identify the natural vectors of Dirofilaria repens Railliet et Henry, entomological samplings were carried out in four sites within the Lazio region, foci of canine subcutaneous dirofilariasis. Collections were made in 2002-2003 by means of dog-baited and miniature Centers for Disease Control and Prevention traps as well as on humans. Microscopy identified 1576 attracted mosquito females as belonging to six species, but molecular diagnostics detected filarial DNA only in Culex pipiens L. and Aedes albopictus (Skuse, 1894). Dirofilaria immitis Leidy DNA, D. repens DNA, or both were found in the head and thorax of both mosquitoes. The simultaneous presence of vectors showing diurnal and nocturnal activity patterns is of concern for animal and human health. The finding of D. immitis DNA in mosquitoes in areas where only D. repens was been recovered in dogs also demonstrates that this filarial parasite circulates among carnivores (wild or domesticated pets).

Microbial Analysis of Bite Marks by Sequence Comparison of Streptococcal DNA

PubMed Central

Kennedy, Darnell M.; Stanton, Jo-Ann L.; García, José A.; Mason, Chris; Rand, Christy J.; Kieser, Jules A.; Tompkins, Geoffrey R.

2012-01-01

Bite mark injuries often feature in violent crimes. Conventional morphometric methods for the forensic analysis of bite marks involve elements of subjective interpretation that threaten the credibility of this field. Human DNA recovered from bite marks has the highest evidentiary value, however recovery can be compromised by salivary components. This study assessed the feasibility of matching bacterial DNA sequences amplified from experimental bite marks to those obtained from the teeth responsible, with the aim of evaluating the capability of three genomic regions of streptococcal DNA to discriminate between participant samples. Bite mark and teeth swabs were collected from 16 participants. Bacterial DNA was extracted to provide the template for PCR primers specific for streptococcal 16S ribosomal RNA (16S rRNA) gene, 16S–23S intergenic spacer (ITS) and RNA polymerase beta subunit (rpoB). High throughput sequencing (GS FLX 454), followed by stringent quality filtering, generated reads from bite marks for comparison to those generated from teeth samples. For all three regions, the greatest overlaps of identical reads were between bite mark samples and the corresponding teeth samples. The average proportions of reads identical between bite mark and corresponding teeth samples were 0.31, 0.41 and 0.31, and for non-corresponding samples were 0.11, 0.20 and 0.016, for 16S rRNA, ITS and rpoB, respectively. The probabilities of correctly distinguishing matching and non-matching teeth samples were 0.92 for ITS, 0.99 for 16S rRNA and 1.0 for rpoB. These findings strongly support the tenet that bacterial DNA amplified from bite marks and teeth can provide corroborating information in the identification of assailants. PMID:23284761

Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis

PubMed Central

Zhao, Hongjuan; Hastie, Trevor; Whitfield, Michael L; Børresen-Dale, Anne-Lise; Jeffrey, Stefanie S

2002-01-01

Background T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. Results Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3–3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. Conclusion T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays. PMID:12445333

Analysis of 4-aminobiphenyl-DNA adducts in human urinary bladder and lung by alkaline hydrolysis and negative ion gas chromatography-mass spectrometry.

PubMed Central

Lin, D; Lay, J O; Bryant, M S; Malaveille, C; Friesen, M; Bartsch, H; Lang, N P; Kadlubar, F F

1994-01-01

Analysis of carcinogen-DNA adducts has been regarded as a useful means of assessing human exposure to chemical carcinogens. We have established a method for quantitation of 4-aminobiphenyl (4-ABP)-DNA adducts by alkaline hydrolysis and gas chromatography with negative ion chemical ionization mass spectrometry (GC-NICI-MS). Aliquots of DNA (typically 100 micrograms/ml) were spiked with an internal standard, d9-4-ABP, and were hydrolyzed in 0.05 N NaOH at 130 degrees C overnight. The liberated 4-ABP was extracted with hexane and derivatized using pentafluoropropionic anhydride in trimethylamine for 30 min at room temperature prior to GC-NICI-MS. With in vitro [3H]N-hydroxy-4-ABP modified DNA standards, we observed 59 +/- 7% (n = 9) recovery of the 4-ABP and a linear correlation between hydrolyzed 4-ABP and the adduct levels ranging from about 1 in 10(8) to 1 in 10(4) nucleotides (r = 0.999, n = 9). The method was further validated by comparison of the results with that obtained by the 32P-postlabeling method. There was excellent agreement (r = 0.994, p < 0.001) between the two methods for quantitation of the adduct in eight samples of Salmonella typhimurium DNA treated with 4-ABP and rat liver S9, although the 32P-postlabeling method gave slightly higher values. The DNA adducts in 11 human lung and 8 urinary bladder mucosa specimens were then determined by our GC-NICI-MS method. The adduct levels were found to be < 0.32 to 49.5 adducts per 10(8) nucleotides in the lungs and < 0.32 to 3.94 adducts per 10(8) nucleotides in the bladder samples.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 4. A Figure 4. B PMID:7889831

Improved detection of endoparasite DNA in soil sample PCR by the use of anti-inhibitory substances.

PubMed

Krämer, F; Vollrath, T; Schnieder, T; Epe, C

2002-09-26

Although there have been numerous microbial examinations of soil for the presence of human pathogenic developmental parasite stages of Ancylostoma caninum and Toxocara canis, molecular techniques (e.g. DNA extraction, purification and subsequent PCR) have scarcely been applied. Here, DNA preparations of soil samples artificially contaminated with genomic DNA or parasite eggs were examined by PCR. A. caninum and T. canis-specific primers based on the ITS-2 sequence were used for amplification. After the sheer DNA preparation a high content of PCR-interfering substances was still detectable. Subsequently, two different inhibitors of PCR-interfering agents (GeneReleaser, Bioventures Inc. and Maximator, Connex GmbH) were compared in PCR. Both substances increased PCR sensitivity greatly. However, comparison of the increase in sensitivity achieved with the two compounds demonstrated the superiority of Maximator, which enhanced sensitivity to the point of permitting positive detection of a single A. caninum egg and three T. canis eggs in a soil sample. This degree of sensitivity could not be achieved with GeneReleaser for either parasite Furthermore, Maximator not only increased sensitivity; it also cost less, required less time and had a lower risk of contamination. Future applications of molecular methods in epidemiological examinations of soil samples are discussed/elaborated.

Use of hybridot assay to screen for BK and JC polyomaviruses in non-immunosuppressed patients.

PubMed Central

Cobb, J J; Wickenden, C; Snell, M E; Hulme, B; Malcolm, A D; Coleman, D V

1987-01-01

Urine samples from 50 patients attending a genitourinary outpatient clinic and from 13 renal allograft recipients were investigated for evidence of infection with human BK and JC polyomaviruses using cytology and a new DNA hybridot assay. Forty four per cent of samples from the renal allograft recipients were positive by cytology and 75% by DNA hybridisation, indicating that hybridot assay is more sensitive than cytological screening. BK and JC viral DNA was found in 20% of the patients attending the genitourinary clinic, showing infection with BK virus and JC virus in a group of patients with clinical conditions not normally associated with immunological deficiency-a finding that has not been reported before. Images Figure PMID:3040812

DNA methylation age of human tissues and cell types

PubMed Central

2013-01-01

Background It is not yet known whether DNA methylation levels can be used to accurately predict age across a broad spectrum of human tissues and cell types, nor whether the resulting age prediction is a biologically meaningful measure. Results I developed a multi-tissue predictor of age that allows one to estimate the DNA methylation age of most tissues and cell types. The predictor, which is freely available, was developed using 8,000 samples from 82 Illumina DNA methylation array datasets, encompassing 51 healthy tissues and cell types. I found that DNA methylation age has the following properties: first, it is close to zero for embryonic and induced pluripotent stem cells; second, it correlates with cell passage number; third, it gives rise to a highly heritable measure of age acceleration; and, fourth, it is applicable to chimpanzee tissues. Analysis of 6,000 cancer samples from 32 datasets showed that all of the considered 20 cancer types exhibit significant age acceleration, with an average of 36 years. Low age-acceleration of cancer tissue is associated with a high number of somatic mutations and TP53 mutations, while mutations in steroid receptors greatly accelerate DNA methylation age in breast cancer. Finally, I characterize the 353 CpG sites that together form an aging clock in terms of chromatin states and tissue variance. Conclusions I propose that DNA methylation age measures the cumulative effect of an epigenetic maintenance system. This novel epigenetic clock can be used to address a host of questions in developmental biology, cancer and aging research. PMID:24138928

8-oxo-7,8-dihydroguanine level - the DNA oxidative stress marker - recognized by fluorescence image analysis in sporadic uterine adenocarcinomas in women.

PubMed

Postawski, Krzysztof; Przadka-Rabaniuk, Dorota; Piersiak, Tomasz

2013-01-01

In the case of carcinogenesis in human endometrium no information exists on tissue concentration of 8-oxo-7,8-dihydroguanine, the DNA oxidative stress marker This was the main reason to undertake the investigation of this DNA modification in human uterine estrogen-dependent tissue cancers. In order to estimate the level of oxidative damage, 8-oxo-7,8-dihydroguanine was determined directly in cells of tissue microscope slides using OxyDNA Assay Kit, Fluorometric. Cells were investigated under confocal microscope. Images of individual cells were captured by computer-interfaced digital photography and analyzed for fluorescence intensities (continuous inverted 8-bit gray-scale = 0 [black]-255 [white]). Fluorescence scores were calculated for each of 13 normal endometrial samples and 31 uterine adenocarcinoma specimens. Finally the level of the oxidative stress marker was also analyzed according to histological and clinical features of the neoplasms. The obtained data revealed that: 8-oxo-7,8-dihydroguanine levels were higher in uterine adenocarcinomas than in normal endometrial samples (48,32 vs. 38,64; p<0,001); in contrast to normal endometrium there was no correlation between age and DNA oxidative modification content in uterine cancer; highest mean fluorescence intensity was recognized in G2 endometrial adenocarcinomas; level of 8-oxo-7,8-dihydroguanine does not depend on Body Mass Index (BMI) and cancer uterine wall infiltration or tumor FIGO stage. Our study indicates that accumulation of the oxidized DNA base may contribute to the development of endometrial neoplasia, however oxidative DNA damage does not seem to increase with tumor progression.

High density of Leishmania major and rarity of other mammals' Leishmania in zoonotic cutaneous leishmaniasis foci, Iran.

PubMed

Bordbar, Ali; Parvizi, Parviz

2014-03-01

Only Leishmania major is well known as a causative agent of zoonotic cutaneous leishmaniasis (ZCL) in Iran. Our objective was to find Leishmania parasites circulating in reservoir hosts, sand flies and human simultaneously. Sand flies, rodents and prepared smears of humans were sampled. DNA of Leishmania parasites was extracted, and two fragments of ITS-rDNA gene amplified by PCR. RFLP and sequencing were employed to identify Leishmania parasites. Leishmania major and L. turanica were identified unequivocally by targeting and sequencing ITS-rDNA from humans, rodents and sand flies. The new Leishmania species close to gerbilli (GenBank Accession Nos. EF413076; EF413087) was discovered only in sand flies. Based on parasite detection of ITS-rDNA in main and potential reservoir hosts and vectors and humans, we conclude that at least two Leishmania species are common in the Turkmen Sahra ZCL focus. Phylogenetic analysis proved that the new Leishmania is closely related to Leishmania mammal parasites (Leishmania major, Leishmania turanica, Leishmania gerbilli). Its role as a principal agent of ZCL is unknown because it was found only in sand flies. Our findings shed new light on the transmission cycles of several Leishmania parasites in sand flies, reservoir hosts and humans. © 2014 John Wiley & Sons Ltd.

Mitochondrial genome diversity in the Tubalar, Even, and Ulchi: contribution to prehistory of native Siberians and their affinities to Native Americans.

PubMed

Sukernik, Rem I; Volodko, Natalia V; Mazunin, Ilya O; Eltsov, Nikolai P; Dryomov, Stanislav V; Starikovskaya, Elena B

2012-05-01

To fill remaining gaps in mitochondrial DNA diversity in the least surveyed eastern and western flanks of Siberia, 391 mtDNA samples (144 Tubalar from Altai, 87 Even from northeastern Siberia, and 160 Ulchi from the Russian Far East) were characterized via high-resolution restriction fragment length polymorphism/single nucleotide polymorphisms analysis. The subhaplogroup structure was extended through complete sequencing of 67 mtDNA samples selected from these and other related native Siberians. Specifically, we have focused on the evolutionary histories of the derivatives of M and N haplogroups, putatively reflecting different phases of settling Siberia by early modern humans. Population history and phylogeography of the resulting mtDNA genomes, combined with those from previously published data sets, revealed a wide range of tribal- and region-specific mtDNA haplotypes that emerged or diversified in Siberia before or after the last glacial maximum, ∼18 kya. Spatial distribution and ages of the "east" and "west" Eurasian mtDNA haploclusters suggest that anatomically modern humans that originally colonized Altai derived from macrohaplogroup N and came from Southwest Asia around 38,000 years ago. The derivatives of macrohaplogroup M, which largely emerged or diversified within the Russian Far East, came along with subsequent migrations to West Siberia millennia later. The last glacial maximum played a critical role in the timing and character of the settlement of the Siberian subcontinent. Copyright © 2012 Wiley Periodicals, Inc.

Recurrent DNA inversion rearrangements in the human genome

PubMed Central

Flores, Margarita; Morales, Lucía; Gonzaga-Jauregui, Claudia; Domínguez-Vidaña, Rocío; Zepeda, Cinthya; Yañez, Omar; Gutiérrez, María; Lemus, Tzitziki; Valle, David; Avila, Ma. Carmen; Blanco, Daniel; Medina-Ruiz, Sofía; Meza, Karla; Ayala, Erandi; García, Delfino; Bustos, Patricia; González, Víctor; Girard, Lourdes; Tusie-Luna, Teresa; Dávila, Guillermo; Palacios, Rafael

2007-01-01

Several lines of evidence suggest that reiterated sequences in the human genome are targets for nonallelic homologous recombination (NAHR), which facilitates genomic rearrangements. We have used a PCR-based approach to identify breakpoint regions of rearranged structures in the human genome. In particular, we have identified intrachromosomal identical repeats that are located in reverse orientation, which may lead to chromosomal inversions. A bioinformatic workflow pathway to select appropriate regions for analysis was developed. Three such regions overlapping with known human genes, located on chromosomes 3, 15, and 19, were analyzed. The relative proportion of wild-type to rearranged structures was determined in DNA samples from blood obtained from different, unrelated individuals. The results obtained indicate that recurrent genomic rearrangements occur at relatively high frequency in somatic cells. Interestingly, the rearrangements studied were significantly more abundant in adults than in newborn individuals, suggesting that such DNA rearrangements might start to appear during embryogenesis or fetal life and continue to accumulate after birth. The relevance of our results in regard to human genomic variation is discussed. PMID:17389356

Biomolecular identification of ancient Mycobacterium tuberculosis complex DNA in human remains from Britain and continental Europe.

PubMed

Müller, Romy; Roberts, Charlotte A; Brown, Terence A

2014-02-01

Tuberculosis is known to have afflicted humans throughout history and re-emerged towards the end of the 20th century, to an extent that it was declared a global emergency in 1993. The aim of this study was to apply a rigorous analytical regime to the detection of Mycobacterium tuberculosis complex (MTBC) DNA in 77 bone and tooth samples from 70 individuals from Britain and continental Europe, spanning the 1st-19th centuries AD. We performed the work in dedicated ancient DNA facilities designed to prevent all types of modern contamination, we checked the authenticity of all products obtained by the polymerase chain reaction, and we based our conclusions on up to four replicate experiments for each sample, some carried out in an independent laboratory. We identified 12 samples that, according to our strict criteria, gave definite evidence for the presence of MTBC DNA, and another 22 that we classified as "probable" or "possible." None of the definite samples came from vertebrae displaying lesions associated with TB. Instead, eight were from ribs displaying visceral new bone formation, one was a tooth from a skeleton with rib lesions, one was taken from a skeleton with endocranial lesions, one from an individual with lesions to the sacrum and sacroiliac joint and the last was from an individual with no lesions indicative of TB or possible TB. Our results add to information on the past temporal and geographical distribution of TB and affirm the suitability of ribs for studying ancient TB. Copyright © 2013 Wiley Periodicals, Inc.

Tracing Males From Different Continents by Genotyping JC Polyomavirus in DNA From Semen Samples.

PubMed

Rotondo, John Charles; Candian, Tommaso; Selvatici, Rita; Mazzoni, Elisa; Bonaccorsi, Gloria; Greco, Pantaleo; Tognon, Mauro; Martini, Fernanda

2017-05-01

The human JC polyomavirus (JCPyV) is an ubiquitous viral agent infecting approximately 60% of humans. Recently, JCPyV sequences have been detected in semen samples. The aim of this investigation was to test whether semen JCPyV genotyping can be employed to trace the origin continent of males. Semen DNA samples (n = 170) from males of different Continents were investigated by PCR for the polymorphic JCPyV viral capsid protein 1 (VP1) sequences, followed by DNA sequencing. JCPyV sequences were detected with an overall prevalence of 27.6% (47/170). DNA sequencing revealed that European males carried JCPyV types 1A (71.4%), 4 (11.4%), 2B (2.9%), 2D1 (2.9%), and 3A (2.9%). Asians JCPyV type 2D1 (66.7%) and Africans JCPyV types 3A (33.3%) and 1A (33.3%). In 10.6% of males, two different JCPyV genotypes were detected, suggesting that the second JCPyV genotype was acquired in the destination country. This study indicates that the majority of semen samples found to be JCPyV-positive, were infected with the JCPyV genotype found in the geographic area of male origin. Therefore, semen JCPyV genotyping could be employed to trace the origin continent of males. Our findings could be applied to forensic investigations, in case of for instance sexual crimes. Indeed, JCPyV genotyping should enable investigators to make additional detailed profiling of the offender. J. Cell. Physiol. 232: 982-985, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

DNA methylation and targeted sequencing of methyltransferases family genes in canine acute myeloid leukaemia, modelling human myeloid leukaemia.

PubMed

Bronzini, I; Aresu, L; Paganin, M; Marchioretto, L; Comazzi, S; Cian, F; Riondato, F; Marconato, L; Martini, V; Te Kronnie, G

2017-09-01

Tumours shows aberrant DNA methylation patterns, being hypermethylated or hypomethylated compared with normal tissues. In human acute myeloid leukaemia (hAML) mutations in DNA methyltransferase (DNMT3A) are associated to a more aggressive tumour behaviour. As AML is lethal in dogs, we defined global DNA methylation content, and screened the C-terminal domain of DNMT3 family of genes for sequence variants in 39 canine acute myeloid leukaemia (cAML) cases. A heterogeneous pattern of DNA methylation was found among cAML samples, with subsets of cases being hypermethylated or hypomethylated compared with healthy controls; four recurrent single nucleotide variations (SNVs) were found in DNMT3L gene. Although SNVs were not directly correlated to whole genome DNA methylation levels, all hypomethylated cAML cases were homozygous for the deleterious mutation at p.Arg222Trp. This study contributes to understand genetic modifications of cAML, leading up to studies that will elucidate the role of methylome alterations in the pathogenesis of AML in dogs. © 2016 John Wiley & Sons Ltd.

Double-strand DNA-templated formation of copper nanoparticles as fluorescent probe for label-free aptamer sensor.

PubMed

Zhou, Zhixue; Du, Yan; Dong, Shaojun

2011-07-01

Double-strand DNA (dsDNA) can act as an efficient template for the formation of copper nanoparticles (Cu NPs) at low concentration of CuSO(4), and the formed Cu NPs have excellent fluorescence, whereas a single-strand DNA (ssDNA) template does not support Cu NPs' formation. This property of dsDNA-Cu NPs makes it suitable for DNA sensing. However, exploration of dsDNA-Cu NPs applied in biological analysis is still at an early stage. In this regard, we report herein for the first time a sensitive, cost-effective, and simple aptamer sensor (aptasensor) using dsDNA-Cu NPs as fluorescent probe. The design consists of a dsDNA with reporter DNA (here, aptamer) as template for the formation of Cu NPs, and the formed dsDNA-Cu NPs show high fluorescence. Using adenosine triphosphate (ATP) as a model analyte, the introduction of ATP triggers the structure switching of reporter DNA to form aptamer-ATP complex, causing the destruction of the double helix and thus no formation of the Cu NPs, resulting in low fluorescence. The preferable linear range (0.05-500 μM), sensitivity (LOD 28 nM), and simplicity for the detection of ATP indicate that dsDNA-Cu NPs may have great prospects in the field of biological analysis. We also use this novel fluorescent probe to determine ATP in 1% human serum and human adenocarcinoma HeLa cells. The dsDNA-Cu NPs probes provide recovery of 104-108% in 1% human serum and a prominent fluorescent signal is obtained in cellular ATP assay, revealing the practicality of using dsDNA-Cu NPs for the determination of ATP in real samples. Besides, this design is simply based on nucleic acid hybridization, so it can be generally applied to other aptamers for label-free detection of a broad range of analytes. Successful detection of cocaine with detection limit of 0.1 μM demonstrates its potential to be a general method.

Prevalence, identification by a DNA microarray-based assay of human and food isolates Listeria spp. from Tunisia.

PubMed

Hmaïed, F; Helel, S; Le Berre, V; François, J-M; Leclercq, A; Lecuit, M; Smaoui, H; Kechrid, A; Boudabous, A; Barkallah, I

2014-02-01

We aimed at evaluating the prevalence of Listeria species isolated from food samples and characterizing food and human cases isolates. Between 2005 and 2007, one hundred food samples collected in the markets of Tunis were analysed in our study. Five strains of Listeria monocytogenes responsible for human listeriosis isolated in hospital of Tunis were included. Multiplex PCR serogrouping and pulsed field gel electrophoresis (PFGE) applying the enzyme AscI and ApaI were used for the characterization of isolates of L. monocytogenes. We have developed a rapid microarray-based assay to a reliable discrimination of species within the Listeria genus. The prevalence of Listeria spp. in food samples was estimated at 14% by using classical biochemical identification. Two samples were assigned to L. monocytogenes and 12 to L. innocua. DNA microarray allowed unambiguous identification of Listeria species. Our results obtained by microarray-based assay were in accordance with the biochemical identification. The two food L. monocytogenes isolates were assigned to the PCR serogroup IIa (serovar 1/2a). Whereas human L. monocytogenes isolates were of PCR serogroup IVb, (serovars 4b). These isolates present a high similarity in PFGE. Food L. monocytogenes isolates were classified into two different pulsotypes. These pulsotypes were different from that of the five strains responsible for the human cases. We confirmed the presence of Listeria spp. in variety of food samples in Tunis. Increased food and clinical surveillance must be taken into consideration in Tunisia to identify putative infections sources. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

The Fungal Frontier: A Comparative Analysis of Methods Used in the Study of the Human Gut Mycobiome.

PubMed

Huseyin, Chloe E; Rubio, Raul Cabrera; O'Sullivan, Orla; Cotter, Paul D; Scanlan, Pauline D

2017-01-01

The human gut is host to a diverse range of fungal species, collectively referred to as the gut "mycobiome". The gut mycobiome is emerging as an area of considerable research interest due to the potential roles of these fungi in human health and disease. However, there is no consensus as to what the best or most suitable methodologies available are with respect to characterizing the human gut mycobiome. The aim of this study is to provide a comparative analysis of several previously published mycobiome-specific culture-dependent and -independent methodologies, including choice of culture media, incubation conditions (aerobic versus anaerobic), DNA extraction method, primer set and freezing of fecal samples to assess their relative merits and suitability for gut mycobiome analysis. There was no significant effect of media type or aeration on culture-dependent results. However, freezing was found to have a significant effect on fungal viability, with significantly lower fungal numbers recovered from frozen samples. DNA extraction method had a significant effect on DNA yield and quality. However, freezing and extraction method did not have any impact on either α or β diversity. There was also considerable variation in the ability of different fungal-specific primer sets to generate PCR products for subsequent sequence analysis. Through this investigation two DNA extraction methods and one primer set was identified which facilitated the analysis of the mycobiome for all samples in this study. Ultimately, a diverse range of fungal species were recovered using both approaches, with Candida and Saccharomyces identified as the most common fungal species recovered using culture-dependent and culture-independent methods, respectively. As has been apparent from ecological surveys of the bacterial fraction of the gut microbiota, the use of different methodologies can also impact on our understanding of gut mycobiome composition and therefore requires careful consideration. Future research into the gut mycobiome needs to adopt a common strategy to minimize potentially confounding effects of methodological choice and to facilitate comparative analysis of datasets.

DNA viewed as an out-of-equilibrium structure

NASA Astrophysics Data System (ADS)

Provata, A.; Nicolis, C.; Nicolis, G.

2014-05-01

The complexity of the primary structure of human DNA is explored using methods from nonequilibrium statistical mechanics, dynamical systems theory, and information theory. A collection of statistical analyses is performed on the DNA data and the results are compared with sequences derived from different stochastic processes. The use of χ2 tests shows that DNA can not be described as a low order Markov chain of order up to r =6. Although detailed balance seems to hold at the level of a binary alphabet, it fails when all four base pairs are considered, suggesting spatial asymmetry and irreversibility. Furthermore, the block entropy does not increase linearly with the block size, reflecting the long-range nature of the correlations in the human genomic sequences. To probe locally the spatial structure of the chain, we study the exit distances from a specific symbol, the distribution of recurrence distances, and the Hurst exponent, all of which show power law tails and long-range characteristics. These results suggest that human DNA can be viewed as a nonequilibrium structure maintained in its state through interactions with a constantly changing environment. Based solely on the exit distance distribution accounting for the nonequilibrium statistics and using the Monte Carlo rejection sampling method, we construct a model DNA sequence. This method allows us to keep both long- and short-range statistical characteristics of the native DNA data. The model sequence presents the same characteristic exponents as the natural DNA but fails to capture spatial correlations and point-to-point details.

DNA viewed as an out-of-equilibrium structure.

PubMed

Provata, A; Nicolis, C; Nicolis, G

2014-05-01

The complexity of the primary structure of human DNA is explored using methods from nonequilibrium statistical mechanics, dynamical systems theory, and information theory. A collection of statistical analyses is performed on the DNA data and the results are compared with sequences derived from different stochastic processes. The use of χ^{2} tests shows that DNA can not be described as a low order Markov chain of order up to r=6. Although detailed balance seems to hold at the level of a binary alphabet, it fails when all four base pairs are considered, suggesting spatial asymmetry and irreversibility. Furthermore, the block entropy does not increase linearly with the block size, reflecting the long-range nature of the correlations in the human genomic sequences. To probe locally the spatial structure of the chain, we study the exit distances from a specific symbol, the distribution of recurrence distances, and the Hurst exponent, all of which show power law tails and long-range characteristics. These results suggest that human DNA can be viewed as a nonequilibrium structure maintained in its state through interactions with a constantly changing environment. Based solely on the exit distance distribution accounting for the nonequilibrium statistics and using the Monte Carlo rejection sampling method, we construct a model DNA sequence. This method allows us to keep both long- and short-range statistical characteristics of the native DNA data. The model sequence presents the same characteristic exponents as the natural DNA but fails to capture spatial correlations and point-to-point details.

The Fulton County Medical Examiner's experience with the Federal Bureau of Investigation National Missing Person DNA Database Program, 2004-2007.

PubMed

Heninger, Michael; Hanzlick, Randy

2011-03-01

Medical examiners and coroners occasionally encounter unidentified human bodies, which remain unidentified for extended periods. In such cases, when traditional methods of identification have failed or cannot be used, DNA profiling may be used. The Federal Bureau of Investigation has a National Missing Person DNA database (NMPDD) laboratory to which samples may be submitted on such cases and from possible relatives or environments of unidentified decedents. This article describes the experience of the Fulton County Medical Examiner (FCME) in submitting samples to the NMPDD laboratory. A database was established at the FCME to track the submission of samples from unidentified decedents to the NMPDD laboratory for DNA testing along with the results and turnaround times. In December 2004, the FCME inventoried all cases for which samples were available and began to submit them to the NMPDD laboratory for testing. DNA testing and isolation rates, sample type, and turnaround times were tabulated in October 2006 for samples submitted between December 16, 2004 and December 16, 2005. An overall summary of data was also prepared concerning the status of all samples submitted as of April 17, 2007. During the 1-year study period, samples from 77 unidentified decedents were submitted to the laboratory. As of October 2006 (22 months after submission of the first samples and 10 months after submission of the last samples), testing had been completed on 53% of the samples submitted, and 68% of those tested resulted in a mitochondrial DNA profile. Turnaround times ranged from 66 to 557 days, improved with time, and had a mean of 107 days for specimens submitted during the latter part of the study period. As of April 17, 2007, we had submitted samples involving 84 unidentified decedents. Seventy-five percent of the samples have now been tested. Data from the NMPDD laboratory have resulted in 4 identifications by comparison with putative relatives, 4 exclusions, and no cold hits through comparison NMPDD DNA profiles from missing persons. More extensive data are presented in the body of this article. The NMPDD laboratory provides useful and free services to medical examiners, coroners, and law enforcement agencies that require DNA services regarding missing and unidentified persons. Turnaround times have improved. The success of the system in getting cold hits will be heavily dependent on law enforcement filing missing persons reports and submission of reference samples from putative relatives of the decedent. We recommend collecting specimens for DNA analysis early on in the postmortem investigation, submitting samples to the NMPDD laboratory or one of its participating laboratories when traditional methods for identification cannot be used or have failed, not burying bodies until a DNA profile has been obtained, and not cremating unidentified remains.

Alterations of GSH and MDA levels and their association with bee venom-induced DNA damage in human peripheral blood leukocytes.

PubMed

Gajski, Goran; Domijan, Ana-Marija; Garaj-Vrhovac, Vera

2012-07-01

Bee venom (BV) has toxic effects in a variety of cell systems and oxidative stress has been proposed as a possible mechanism of its toxicity. This study investigated the in vitro effect of BV on glutathione (GSH) and malondialdehyde (MDA) levels, and their association with BV-induced DNA strand breaks and oxidative DNA damage in human peripheral blood leukocytes (HPBLs). Blood samples were treated with BV at concentrations ranging from 0.1 to 10 μg/ml over different lengths of time, and DNA damage in HPBLs was monitored with the alkaline and formamidopyrimidine glycoslyase (FPG)-modified comet assays, while GSH and MDA levels were determined in whole blood. Results showed a significant increase in overall DNA damage and FPG-sensitive sites in DNA of HPBLs exposed to BV compared with HPBLs from controls. An increase in DNA damage (assessed with both comet assays) was significantly associated with changes in MDA and GSH levels. When pretreated with N-acetyl-L-cysteine, a source of cysteine for the synthesis of the endogenous antioxidant GSH, a significant reduction of the DNA damaging effects of BV in HPBLs was noted. This suggests that oxidative stress is at least partly responsible for the DNA damaging effects of BV. Copyright © 2012 Wiley Periodicals, Inc.

Detection and quantification of 4-ABP adducts in DNA from bladder cancer patients.

PubMed

Zayas, Beatriz; Stillwell, Sara W; Wishnok, John S; Trudel, Laura J; Skipper, Paul; Yu, Mimi C; Tannenbaum, Steven R; Wogan, Gerald N

2007-02-01

We analyzed bladder DNA from 27 cancer patients for dG-C8-4-aminobiphenyl (dG-C8-ABP) adducts using the liquid chromatography tandem mass spectrometry method with a 700 attomol (1 adduct in 10(9) bases) detection limit. Hemoglobin (Hb) 4-aminobiphenyl (4-ABP) adduct levels were measured by gas chromatography-mass spectrometry. After isolation of dG-C8-ABP by immunoaffinity chromatography and further purification, deuterated (d9) dG-C8-ABP (MW=443 Da) was added to each sample. Structural evidence and adduct quantification were determined by selected reaction monitoring, based on the expected adduct ion [M+H+]+1, at m/z 435 with fragmentation to the product ion at m/z 319, and monitoring of the transition for the internal standard, m/z 444-->328. The method was validated by analysis of DNA (100 microg each) from calf thymus; livers from ABP-treated and untreated rats; human placentas; and TK6 lymphoblastoid cells. Adduct was detected at femtomol levels in DNA from livers of ABP-treated rats and calf thymus, but not in other controls. The method was applied to 41 DNA samples (200 microg each) from 27 human bladders; 28 from tumor and 14 from surrounding non-tumor tissue. Of 27 tissues analyzed, 44% (12) contained 5-80 dG-C8-ABP adducts per 10(9) bases; only 1 out of 27 (4%) contained adduct in both tumor and surrounding tissues. The Hb adduct was detected in samples from all patients, at levels of 12-1960 pg per gram Hb. There was no correlation between levels of DNA and Hb adducts. The presence of DNA adducts in 44% of the subjects and high levels of Hb adducts in these non-smokers indicate environmental sources of exposure to 4-ABP.

The Control Region of Mitochondrial DNA Shows an Unusual CpG and Non-CpG Methylation Pattern

PubMed Central

Bellizzi, Dina; D'Aquila, Patrizia; Scafone, Teresa; Giordano, Marco; Riso, Vincenzo; Riccio, Andrea; Passarino, Giuseppe

2013-01-01

DNA methylation is a common epigenetic modification of the mammalian genome. Conflicting data regarding the possible presence of methylated cytosines within mitochondrial DNA (mtDNA) have been reported. To clarify this point, we analysed the methylation status of mtDNA control region (D-loop) on human and murine DNA samples from blood and cultured cells by bisulphite sequencing and methylated/hydroxymethylated DNA immunoprecipitation assays. We found methylated and hydroxymethylated cytosines in the L-strand of all samples analysed. MtDNA methylation particularly occurs within non-C-phosphate-G (non-CpG) nucleotides, mainly in the promoter region of the heavy strand and in conserved sequence blocks, suggesting its involvement in regulating mtDNA replication and/or transcription. We observed DNA methyltransferases within the mitochondria, but the inactivation of Dnmt1, Dnmt3a, and Dnmt3b in mouse embryonic stem (ES) cells results in a reduction of the CpG methylation, while the non-CpG methylation shows to be not affected. This suggests that D-loop epigenetic modification is only partially established by these enzymes. Our data show that DNA methylation occurs in the mtDNA control region of mammals, not only at symmetrical CpG dinucleotides, typical of nuclear genome, but in a peculiar non-CpG pattern previously reported for plants and fungi. The molecular mechanisms responsible for this pattern remain an open question. PMID:23804556

A cascade signal amplification strategy for sensitive and label-free DNA detection based on Exo III-catalyzed recycling coupled with rolling circle amplification.

PubMed

Liu, Xingti; Xue, Qingwang; Ding, Yongshun; Zhu, Jing; Wang, Lei; Jiang, Wei

2014-06-07

A sensitive and label-free fluorescence assay for DNA detection has been developed based on cascade signal amplification combining exonuclease III (Exo III)-catalyzed recycling with rolling circle amplification. In this assay, probe DNA hybridized with template DNA was coupled onto magnetic nanoparticles to prepare a magnetic bead-probe (MNB-probe)-template complex. The complex could hybridize with the target DNA, which transformed the protruding 3' terminus of template DNA into a blunt end. Exo III could then digest template DNA, liberating the MNB-probe and target DNA. The intact target DNA then hybridized with other templates and released more MNB-probes. The liberated MNB-probe captured the primer, circular DNA and then initiated the rolling circle amplification (RCA) reaction, realizing a cascade signal amplification. Using this cascade amplification strategy, a sensitive DNA detection method was developed which was superior to many existing Exo III-based signal amplification methods. Moreover, N-methyl mesoporphyrin IX, which had a pronounced structural selectivity for the G-quadruplex, was used to combine with the G-quadruplex RCA products and generate a fluorescence signal, avoiding the need for any fluorophore-label probes. The spike and recovery experiments in a human serum sample indicated that our assay also had great potential for DNA detection in real biological samples.

Simultaneous detection, typing and quantitation of oncogenic human papillomavirus by multiplex consensus real-time PCR.

PubMed

Jenkins, Andrew; Allum, Anne-Gry; Strand, Linda; Aakre, Randi Kersten

2013-02-01

A consensus multiplex real-time PCR test (PT13-RT) for the oncogenic human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 is described. The test targets the L1 gene. Analytical sensitivity is between 4 and 400 GU (genomic units) in the presence of 500 ng of human DNA, corresponding to 75,000 human cells. HPV types are grouped into multiplex groups of 3 or 4 resulting in the use of 4 wells per sample and permitting up to 24 samples per run (including controls) in a standard 96-well real-time PCR instrument. False negative results are avoided by (a) measuring sample DNA concentration to control that sufficient cellular material is present and (b) including HPV type 6 as a homologous internal control in order to detect PCR inhibition or competition from other (non-oncogenic) HPV types. Analysis time from refrigerator to report is 8 h, including 2.5 h hands-on time. Relative to the HC2 test, the sensitivity and specificity were respectively 98% and 83%, the lower specificity being attributable to the higher analytical sensitivity of PT13-RT. To assess type determination comparison was made with a reversed line-blot test. Type concordance was high (κ=0.79) with discrepancies occurring mostly in multiple-positive samples. Copyright © 2012 Elsevier B.V. All rights reserved.

Torque Teno Virus in HIV-infected transgender in Surakarta, Indonesia

NASA Astrophysics Data System (ADS)

Hartono; Agung Prasetyo, Afiono; Fanani, Mohammad

2018-05-01

Torque Teno Virus (TTV) is a circular single-stranded DNA virus that may co-infected with human immunodeficiency virus (HIV), especially in the high-risk community e.g. the transgender performing high-riskbehavior. TTV shows an increased viremia in HIV patients and maybe influence the HIV clinical progression. Blood samples collected from transgender performing high-riskbehavior in Surakarta were tested by serological and molecular assays to detect the presence of HIV infection. The blood samples with HIV positive status were then tested by a nested polymerase chain reaction (PCR) to detect the presentation of TTV DNA. The amplified PCR products were molecularly cloned and subjected to sequence analysis. TTV DNA was detected in 40.0% HIV-positive samples. The molecular characterization revealed that the most prevalent was genogroup 3, followed by genogroup 2 and 1, respectively. TTV was detected in HIV-infected transgender performing high-riskbehavior in Surakarta with high infection rate.

Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota.

PubMed

Horng, Katti R; Ganz, Holly H; Eisen, Jonathan A; Marks, Stanley L

2018-01-01

Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25 °C, 4 °C, and -80 °C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on Days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer ( F -value = 6.87, DF  = 3, P  < 0.001), storage temperature ( F -value=1.77, DF  = 3, P  = 0.037), and duration of sample storage ( F -value = 3.68, DF  = 3, P  < 0.001). Changes in bacterial composition were observed in samples stored in -80 °C without buffer, a commonly used method for fecal DNA storage, suggesting that simply freezing samples may be suboptimal for bacterial analysis. Fecal preservation with 70% ethanol and RNAlater closely resembled that of fresh samples, though RNAlater yielded significantly lower DNA concentrations ( DF  = 8.57, P  < 0.001). Although bacterial composition varied with temperature and buffer storage, 70% ethanol was the best method for preserving bacterial DNA in canine feces, yielding the highest DNA concentration and minimal changes in bacterial diversity and composition. The differences observed between samples highlight the need to consider optimized post-collection methods in microbiome research.

Forensic DNA methylation profiling from minimal traces: How low can we go?

PubMed

Naue, Jana; Hoefsloot, Huub C J; Kloosterman, Ate D; Verschure, Pernette J

2018-03-01

Analysis of human DNA methylation (DNAm) can provide additional investigative leads in crime cases, e.g. the type of tissue or body fluid, the chronological age of an individual, and differentiation between identical twins. In contrast to the genetic profile, the DNAm level is not the same in every cell. At the single cell level, DNAm represents a binary event at a defined CpG site (methylated versus non-methylated). The DNAm level from a DNA extract however represents the average level of methylation of the CpG of interest of all molecules in the forensic sample. The variance of DNAm levels between replicates is often attributed to technological issues, i.e. degradation of DNA due to bisulfite treatment, preferential amplification of DNA, and amplification failure. On the other hand, we show that stochastic variations can lead to gross fluctuation in the analysis of methylation levels in samples with low DNA levels. This stochasticity in DNAm results is relevant since low DNA amounts (1pg - 1ng) is rather the norm than the exception when analyzing forensic DNA samples. This study describes a conceptual analysis of DNAm profiling and its dependence on the amount of input DNA. We took a close look at the variation of DNAm analysis due to DNA input and its consequences for different DNAm-based forensic applications. As can be expected, the 95%-confidence interval of measured DNAm becomes narrower with increasing amounts of DNA. We compared this aspect for two different DNAm-based forensic applications: body fluid identification and chronological age determination. Our study shows that DNA amount should be well considered when using DNAm for forensic applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Epigenetic modification of OXT and human sociability.

PubMed

Haas, Brian W; Filkowski, Megan M; Cochran, R Nick; Denison, Lydia; Ishak, Alexandra; Nishitani, Shota; Smith, Alicia K

2016-07-05

Across many mammalian species there exist genetic and biological systems that facilitate the tendency to be social. Oxytocin is a neuropeptide involved in social-approach behaviors in humans and others mammals. Although there exists a large, mounting body of evidence showing that oxytocin signaling genes are associated with human sociability, very little is currently known regarding the way the structural gene for oxytocin (OXT) confers individual differences in human sociability. In this study, we undertook a comprehensive approach to investigate the association between epigenetic modification of OXT via DNA methylation, and overt measures of social processing, including self-report, behavior, and brain function and structure. Genetic data were collected via saliva samples and analyzed to target and quantify DNA methylation across the promoter region of OXT We observed a consistent pattern of results across sociability measures. People that exhibit lower OXT DNA methylation (presumably linked to higher OXT expression) display more secure attachment styles, improved ability to recognize emotional facial expressions, greater superior temporal sulcus activity during two social-cognitive functional MRI tasks, and larger fusiform gyrus gray matter volume than people that exhibit higher OXT DNA methylation. These findings provide empirical evidence that epigenetic modification of OXT is linked to several overt measures of sociability in humans and serve to advance progress in translational social neuroscience research toward a better understanding of the evolutionary and genetic basis of normal and abnormal human sociability.

Application of ion chromatography for the determination of inorganic ions, especially thiocyanates, in human semen samples as biomarkers of environmental tobacco smoke exposure.

PubMed

Demkowska, Ilona; Polkowska, Żaneta; Kiełbratowska, Bogumiła; Namieśnik, Jacek

2010-11-01

Tobacco smoking constitutes a significant source of indoor air pollution. Various chemical compounds that are emitted during tobacco smoking can have a direct cytotoxic effect on spermatozoa by damaging DNA. There is some evidence that tobacco smoking in men could affect male fertility. The goals of this study were to find relationships between thiocyanates (as biomarkers of environmental tobacco smoke exposure) and other inorganic ions in human semen samples and present the effectiveness of the proposed sample preparation procedure combined with ion chromatography technique for the determination of inorganic ions, especially thiocyanates, in human semen samples collected from heavy, moderate, and passive smokers, as well as nonsmoking individuals.

Distinctive toxicity of TiO2 rutile/anatase mixed phase nanoparticles on Caco-2 cells.

PubMed

Gerloff, Kirsten; Fenoglio, Ivana; Carella, Emanuele; Kolling, Julia; Albrecht, Catrin; Boots, Agnes W; Förster, Irmgard; Schins, Roel P F

2012-03-19

Titanium dioxide has a long-standing use as a food additive. Micrometric powders are, e.g., applied as whiteners in confectionary or dairy products. Possible hazards of ingested nanometric TiO(2) particles for humans and the potential influence of varying specific surface area (SSA) are currently under discussion. Five TiO(2)-samples were analyzed for purity, crystallinity, primary particle size, SSA, ζ potential, and aggregation/agglomeration. Their potential to induce cytotoxicity, oxidative stress, and DNA damage was evaluated in human intestinal Caco-2 cells. Only anatase-rutile containing samples, in contrast to the pure anatase samples, induced significant LDH leakage or mild DNA damage (Fpg-comet assay). Evaluation of the metabolic competence of the cells (WST-1 assay) revealed a highly significant correlation between the SSA of the anatase samples and cytotoxicity. The anatase/rutile samples showed higher toxicity per unit surface area than the pure anatase powders. However, none of the samples affected cellular markers of oxidative stress. Our findings suggest that both SSA and crystallinity are critical determinants of TiO(2)-toxicity toward intestinal cells. © 2012 American Chemical Society

Authenticated DNA from Ancient Wood Remains

PubMed Central

LIEPELT, SASCHA; SPERISEN, CHRISTOPH; DEGUILLOUX, MARIE-FRANCE; PETIT, REMY J.; KISSLING, ROY; SPENCER, MATTHEW; DE BEAULIEU, JACQUES-LOUIS; TABERLET, PIERRE; GIELLY, LUDOVIC; ZIEGENHAGEN, BIRGIT

2006-01-01

• Background The reconstruction of biological processes and human activities during the last glacial cycle relies mainly on data from biological remains. Highly abundant tissues, such as wood, are candidates for a genetic analysis of past populations. While well-authenticated DNA has now been recovered from various fossil remains, the final ‘proof’ is still missing for wood, despite some promising studies. • Scope The goal of this study was to determine if ancient wood can be analysed routinely in studies of archaeology and palaeogenetics. An experiment was designed which included blind testing, independent replicates, extensive contamination controls and rigorous statistical tests. Ten samples of ancient wood from major European forest tree genera were analysed with plastid DNA markers. • Conclusions Authentic DNA was retrieved from wood samples up to 1000 years of age. A new tool for real-time vegetation history and archaeology is ready to use. PMID:16987920

Beyond DNA Sequencing in Space: Current and Future Omics Capabilities of the Biomolecule Sequencer Payload

NASA Technical Reports Server (NTRS)

Wallace, Sarah

2017-01-01

Why do we need a DNA sequencer to support the human exploration of space? (A) Operational environmental monitoring; (1) Identification of contaminating microbes, (2) Infectious disease diagnosis, (3) Reduce down mass (sample return for environmental monitoring, crew health, etc.). (B) Research; (1) Human, (2) Animal, (3) Microbes/Cell lines, (4) Plant. (C) Med Ops; (1) Response to countermeasures, (2) Radiation, (3) Real-time analysis can influence medical intervention. (C) Support astrobiology science investigations; (1) Technology superiorly suited to in situ nucleic acid-based life detection, (2) Functional testing for integration into robotics for extraplanetary exploration mission.

Genome-wide single-nucleotide polymorphism arrays demonstrate high fidelity of multiple displacement-based whole-genome amplification.

PubMed

Tzvetkov, Mladen V; Becker, Christian; Kulle, Bettina; Nürnberg, Peter; Brockmöller, Jürgen; Wojnowski, Leszek

2005-02-01

Whole-genome DNA amplification by multiple displacement (MD-WGA) is a promising tool to obtain sufficient DNA amounts from samples of limited quantity. Using Affymetrix' GeneChip Human Mapping 10K Arrays, we investigated the accuracy and allele amplification bias in DNA samples subjected to MD-WGA. We observed an excellent concordance (99.95%) between single-nucleotide polymorphisms (SNPs) called both in the nonamplified and the corresponding amplified DNA. This concordance was only 0.01% lower than the intra-assay reproducibility of the genotyping technique used. However, MD-WGA failed to amplify an estimated 7% of polymorphic loci. Due to the algorithm used to call genotypes, this was detected only for heterozygous loci. We achieved a 4.3-fold reduction of noncalled SNPs by combining the results from two independent MD-WGA reactions. This indicated that inter-reaction variations rather than specific chromosomal loci reduced the efficiency of MD-WGA. Consistently, we detected no regions of reduced amplification, with the exception of several SNPs located near chromosomal ends. Altogether, despite a substantial loss of polymorphic sites, MD-WGA appears to be the current method of choice to amplify genomic DNA for array-based SNP analyses. The number of nonamplified loci can be substantially reduced by amplifying each DNA sample in duplicate.

Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy.

PubMed

Moen, Aina E F; Tannæs, Tone M; Vatn, Simen; Ricanek, Petr; Vatn, Morten Harald; Jahnsen, Jørgen

2016-06-28

Nucleic acid purification methods are of importance when performing microbiota studies and especially when analysing the intestinal microbiota as we here find a wide range of different microbes. Various considerations must be taken to lyse the microbial cell wall of each microbe. In the present article, we compare several tissue lysis steps and commercial purification kits, to achieve a joint RNA and DNA purification protocol for the purpose of investigating the microbiota and the microbiota-host interactions in a single colonic mucosal tissue sample. A further optimised tissue homogenisation and lysis protocol comprising mechanical bead beating, lysis buffer replacement and enzymatic treatment, in combination with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) resulted in efficient and simultaneous purification of microbial and human RNA and DNA from a single mucosal colonic tissue sample. The present work provides a unique possibility to study RNA and DNA from the same mucosal biopsy sample, making a direct comparison between metabolically active microbes and total microbial DNA. The protocol also offers an opportunity to investigate other members of a microbiota such as viruses, fungi and micro-eukaryotes, and moreover the possibility to extract data on microbiota and host interactions from one single mucosal biopsy.

Oligonucleotide and Parylene Surface Coating of Polystyrene and ePTFE for Improved Endothelial Cell Attachment and Hemocompatibility

PubMed Central

Schleicher, Martina; Hansmann, Jan; Elkin, Bentsian; Kluger, Petra J.; Liebscher, Simone; Huber, Agnes J. T.; Fritze, Olaf; Schille, Christine; Müller, Michaela; Schenke-Layland, Katja; Seifert, Martina; Walles, Heike; Wendel, Hans-Peter; Stock, Ulrich A.

2012-01-01

In vivo self-endothelialization by endothelial cell adhesion on cardiovascular implants is highly desirable. DNA-oligonucleotides are an intriguing coating material with nonimmunogenic characteristics and the feasibility of easy and rapid chemical fabrication. The objective of this study was the creation of cell adhesive DNA-oligonucleotide coatings on vascular implant surfaces. DNA-oligonucleotides immobilized by adsorption on parylene (poly(monoaminomethyl-para-xylene)) coated polystyrene and ePTFE were resistant to high shear stress (9.5 N/m2) and human blood serum for up to 96 h. Adhesion of murine endothelial progenitor cells, HUVECs and endothelial cells from human adult saphenous veins as well as viability over a period of 14 days of HUVECs on oligonucleotide coated samples under dynamic culture conditions was significantly enhanced (P < 0.05). Oligonucleotide-coated surfaces revealed low thrombogenicity and excellent hemocompatibility after incubation with human blood. These properties suggest the suitability of immobilization of DNA-oligonucleotides for biofunctionalization of blood vessel substitutes for improved in vivo endothelialization. PMID:22481939

Purification of crime scene DNA extracts using centrifugal filter devices

PubMed Central

2013-01-01

Background The success of forensic DNA analysis is limited by the size, quality and purity of biological evidence found at crime scenes. Sample impurities can inhibit PCR, resulting in partial or negative DNA profiles. Various DNA purification methods are applied to remove impurities, for example, employing centrifugal filter devices. However, irrespective of method, DNA purification leads to DNA loss. Here we evaluate the filter devices Amicon Ultra 30 K and Microsep 30 K with respect to recovery rate and general performance for various types of PCR-inhibitory crime scene samples. Methods Recovery rates for DNA purification using Amicon Ultra 30 K and Microsep 30 K were gathered using quantitative PCR. Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the general performance and inhibitor-removal properties of the two filter devices. Additionally, the outcome of long-term routine casework DNA analysis applying each of the devices was evaluated. Results Applying Microsep 30 K, 14 to 32% of the input DNA was recovered, whereas Amicon Ultra 30 K retained 62 to 70% of the DNA. The improved purity following filter purification counteracted some of this DNA loss, leading to slightly increased electropherogram peak heights for blood on denim (Amicon Ultra 30 K and Microsep 30 K) and saliva on envelope (Amicon Ultra 30 K). Comparing Amicon Ultra 30 K and Microsep 30 K for purification of DNA extracts from mock crime scene samples, the former generated significantly higher peak heights for rape case samples (P-values <0.01) and for hairs (P-values <0.036). In long-term routine use of the two filter devices, DNA extracts purified with Amicon Ultra 30 K were considerably less PCR-inhibitory in Quantifiler Human qPCR analysis compared to Microsep 30 K. Conclusions Amicon Ultra 30 K performed better than Microsep 30 K due to higher DNA recovery and more efficient removal of PCR-inhibitory substances. The different performances of the filter devices are likely caused by the quality of the filters and plastic wares, for example, their DNA binding properties. DNA purification using centrifugal filter devices can be necessary for successful DNA profiling of impure crime scene samples and for consistency between different PCR-based analysis systems, such as quantification and STR analysis. In order to maximize the possibility to obtain complete STR DNA profiles and to create an efficient workflow, the level of DNA purification applied should be correlated to the inhibitor-tolerance of the STR analysis system used. PMID:23618387

Rapid Detection and Identification of a Pathogen's DNA Using Phi29 DNA Polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Y.; Dunn, J.; Gao, S.

2008-10-31

Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing asmore » little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.« less

Subclinical Reactivation and Shed of Infectious Varicella Zoster Virus in Saliva of Astronauts

NASA Technical Reports Server (NTRS)

Cohrs, Randall J.; Mehta, Satish K.; Schmid, D. Scott; Gilden, Donald H.; Pierson, Duane L.

2007-01-01

We have previously detected VZV in healthy astronauts both during spaceflight and shortly after landing. Herein, we show that VZV shed in seropositive astronauts is infectious. A total of 40 saliva samples were obtained from each of the 3 astronauts. From each astronaut, 14 samples were taken 109 to 133 days before liftoff, 1 sample was taken every day during 12 days in space, and one sample was taken for 14 consecutive days beginning the second day after landing. Quantitative PCR was used to detect VZV DNA in saliva. None of 42 preflight saliva samples contained VZV DNA. VZV DNA was detected in saliva from 2 of 3 astronauts. In 1 astronaut, 6 of 12 samples obtained during space flight contained 120 to 2,500 copies of VZV DNA per ml; after landing, 1250 copies of VZV DNA were present on day 2, 45 copies on day 3, and 110 copies on day 5. All samples taken 6 to 15 days after touchdown were negative for VZV DNA. In the second astronaut, 5 of 12 samples obtained during space flight contained 18 to 650 copies of VZV DNA per ml; after landing, 560 copies of VZV DNA were present in saliva on day 2, 340 copies on day 4, 45 copies on day 5, and 23 copes on day 6. All samples taken 7 to 15 days after touchdown were negative for VZV DNA. Saliva taken 2 to 6 days after landing from all 3 astronauts was cultured on human fetal lung cells. After one subcultivation, a cytopathic effect developed in cultures inoculated with saliva from the two astronauts whose saliva contained VZV DNA. Both PCR and immunostaining identified the isolates to be VZV and not HSV-1. Importantly, the astronaut in whom no VZV was detected had a history of zoster 9 years earlier. It is possible that a boost in cell-mediated immunity to VZV which is known to develop after zoster protected him from subclinical reactivation. The genotype of the two VZV isolates was determined by VZV ORF22-based PCR/sequencing along with FRET-based PCR assays that target specific nucleotide polymorphisms. Both VZV isolates were found to be the European genotype which also contained a rare MspI restriction enodnuclease site in VZV ORF62 at position 107,252. These findings extend our previous demonstration of VZV DNA in saliva of astronauts by showing that infectious VZV is also present. Thus, like HSV-1 and HSV-2, VZV can reactivate and shed infectious virus in the absence of clinical disease.

Stress-induced subclinical reactivation of varicella zoster virus in astronauts

NASA Technical Reports Server (NTRS)

Mehta, Satish K.; Cohrs, Randall J.; Forghani, Bagher; Zerbe, Gary; Gilden, Donald H.; Pierson, Duane L.

2004-01-01

Varicella zoster virus (VZV) becomes latent in human ganglia after primary infection. VZV reactivation occurs primarily in elderly individuals, organ transplant recipients, and patients with cancer and AIDS, correlating with a specific decline in cell-mediated immunity to the virus. VZV can also reactivate after surgical stress. The unexpected occurrence of thoracic zoster 2 days before space flight in a 47-year-old healthy astronaut from a pool of 81 physically fit astronauts prompted our search for VZV reactivation during times of stress to determine whether VZV can also reactivate after non-surgical stress. We examined total DNA extracted from 312 saliva samples of eight astronauts before, during, and after space flight for VZV DNA by polymerase chain reaction: 112 samples were obtained 234-265 days before flight, 84 samples on days 2 through 13 of space flight, and 116 samples on days 1 through 15 after flight. Before space flight, only one of the 112 saliva samples from a single astronaut was positive for VZV DNA. In contrast, during and after space flight, 61 of 200 (30%) saliva samples were positive in all eight astronauts. No VZV DNA was detected in any of 88 saliva samples from 10 healthy control subjects. These results indicate that VZV can reactivate subclinically in healthy individuals after non-surgical stress. Copyright 2004 Wiley-Liss, Inc.

Automation and validation of DNA-banking systems.

PubMed

Thornton, Melissa; Gladwin, Amanda; Payne, Robin; Moore, Rachael; Cresswell, Carl; McKechnie, Douglas; Kelly, Steve; March, Ruth

2005-10-15

DNA banking is one of the central capabilities on which modern genetic research rests. The DNA-banking system plays an essential role in the flow of genetic data from patients and genetics researchers to the application of genetic research in the clinic. Until relatively recently, large collections of DNA samples were not common in human genetics. Now, collections of hundreds of thousands of samples are common in academic institutions and private companies. Automation of DNA banking can dramatically increase throughput, eliminate manual errors and improve the productivity of genetics research. An increased emphasis on pharmacogenetics and personalized medicine has highlighted the need for genetics laboratories to operate within the principles of a recognized quality system such as good laboratory practice (GLP). Automated systems are suitable for such laboratories but require a level of validation that might be unfamiliar to many genetics researchers. In this article, we use the AstraZeneca automated DNA archive and reformatting system (DART) as a case study of how such a system can be successfully developed and validated within the principles of GLP.

Concordance study between the ParaDNA® Intelligence Test, a rapid DNA profiling assay, and a conventional STR typing kit (AmpFlSTR® SGM Plus®).

PubMed

Ball, G; Dawnay, N; Stafford-Allen, B; Panasiuk, M; Rendell, P; Blackman, S; Duxbury, N; Wells, S

2015-05-01

The ParaDNA® Intelligence Test enables STR profiling directly from human biological samples and evidence items collected from crime scene in 75min. Designed for non-expert use this system allows DNA information to be available to investigators before it would typically be available from a laboratory. The ParaDNA Intelligence Test system amplifies D3S1358, D8S119, D16S539, D18S1358 and TH01 STR loci and the gender typing locus amelogenin and detects the alleles present with HyBeacon® probes. Individual DNA samples from 381 UK Caucasian individuals were analysed using AmpFlSTR® SGM Plus® and the ParaDNA Intelligence Test with the derived STR profiles compared. Here we describe the high level of concordance demonstrated between the two systems and discuss this with reference to allele frequencies and the discriminatory power offered by the ParaDNA Intelligence Test. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Detection of Helicobacter spp. DNA in the oral cavity of dogs.

PubMed

Recordati, Camilla; Gualdi, Valentina; Tosi, Sabrina; Facchini, Roberto Vailati; Pengo, Graziano; Luini, Mario; Simpson, Kenneth W; Scanziani, Eugenio

2007-01-31

The mode of acquisition of gastric Helicobacter spp. infection in dogs has not been determined. It is suspected that oral-oral and faecal-oral transmission may be involved. The present study sought to determine if Helicobacter spp. DNA is present in the oral cavity of healthy and vomiting dogs. Thirty-eight pet dogs (27 vomiting and 11 clinically healthy) were studied. The presence of Helicobacter spp. was determined by single and nested PCR evaluation of DNA extracted from saliva, dental plaque and gastric biopsy samples. Helicobacter spp. DNA was detected by nested PCR in 36 (94.7%) gastric biopsies, 17 (44.7%) dental plaque and 19 (50%) saliva samples out of the 38 dogs examined. Overall 27 (71.1%) dogs screened by nested PCR were found to harbour Helicobacter spp. DNA in the oral cavity (dental plaque and/or saliva). There was no significant difference in the prevalence of Helicobacter spp. DNA in the oral cavity of vomiting and healthy dogs, and the time from vomiting to oral sampling did not have significant impact. This study confirms the high prevalence of gastric Helicobacter spp. infection in dogs, and reveals that Helicobacter spp. DNA is detectable in the oral cavity of over 70% of dogs. These findings support the possibility of oral-oral transmission between dogs and that the canine oral cavity may act as source of non-pylori Helicobacter spp. infection for humans.