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1

Assessment of DNA degradation and the genotyping success of highly degraded samples.  

PubMed

DNA becomes progressively more fragmented as biological tissue degrades resulting in decreasing ability to gain a complete DNA profile. Successful identification of samples exhibiting very high levels of DNA degradation may be complicated by presenting in minute quantities. The industry standard method for human DNA identification utilising short tandem repeats (STR) may produce partial or no DNA profile with such samples. We report a comparative study of genotyping using STRs, mini-STRs and single nucleotide polymorphisms (SNPs) with template at different levels of degradation in varying amounts. Two methods of assessing quantity and quality of a DNA sample prior to genotyping were investigated. The QIAxcel capillary gel electrophoresis system provided a rapid, cost effective screening method for assessing sample quality. A real-time quantitative PCR (qPCR) assay was able to simultaneously quantify total human DNA, male DNA, DNA degradation and PCR inhibition. The extent of DNA degradation could be assessed with reasonable accuracy to 62.5 pg and genomic targets could be quantified to a lower limit of 15.6 pg. The qPCR assay was able to detect male DNA to a lower limit of 20 pg in a 1:1,000 background of female DNA. By considering the amount of DNA and the degradation ratio of a sample, a general prediction of genotyping success using AmpFlSTR® Profiler Plus®, MiniFiler™ kits and SNP analysis can be made. The results indicate mini-STRs and SNP markers are usually more successful in typing degraded samples and in cases of extreme DNA degradation (?200 bp) and template amounts below 250 pg, mini-STR and SNP analysis yielded significantly more complete profiles and lower match probabilities than corresponding STR profiles. PMID:20419381

Hughes-Stamm, Sheree R; Ashton, Kevin J; van Daal, Angela

2011-05-01

2

Development of a real-time method to detect DNA degradation in forensic samples.  

PubMed

Knowledge of the degradation state of evidentiary DNA samples would allow selection of the appropriate analysis method (standard short tandem repeats [STRs] vs. mini STRs vs. mtDNA). This article describes the development of a Plexor® technology/real-time PCR DNA degradation detection assay, which uses a common forward primer and two reverse primers (different fluorophores) to generate two Alu amplicons (63 and 246 bp). This very sensitive assay was optimized for reaction volume, cycle number, anneal/extend time, and temperature. Using DNA samples degraded with DNaseI, the ratio of the concentration of the short amplicon to the concentration of the long amplicon (degradation ratio) was increased versus time of degradation. Experiments were performed on a variety of environmentally degraded samples (age, sunlight, heat) and with seven commonly encountered forensic inhibitors. The degradation ratio was found to predict the observed loss of larger STR loci seen in the analysis of comprised samples. PMID:22150029

Nicklas, Janice A; Noreault-Conti, Trisha; Buel, Eric

2012-03-01

3

Comparison of two whole genome amplification methods for STR genotyping of LCN and degraded DNA samples  

Microsoft Academic Search

The analysis of LCN or highly degraded DNA samples presents a challenge for forensic science. Improving the quantity and\\/or quality of samples would greatly increase the profiling success rate from LCN and degraded samples. Whole genome amplification (WGA) is one method that has such potential. Two commercially available WGA kits, GenomePlex and GenomiPhi, were investigated for use on LCN and

Kaye N. Ballantyne; Roland A. H. van Oorschot; R. John Mitchell

2007-01-01

4

Capillary electrophoresis of miniSTR markers to genotype highly degraded DNA samples.  

PubMed

The amplification of short tandem repeat (STR) markers throughout the human nuclear DNA genome are used to associate crime scene evidence to the perpetrator's profile in criminal investigations. For highly challenged or compromised materials such as stains exposed to the elements, skeletal remains from missing persons cases, or fragmented and degraded samples from mass disasters, obtaining a full STR profile may be difficult if not impossible. With the introduction of short amplicon STR or "miniSTR" typing, it is possible to obtain STR genetic information from highly challenged samples without the need to sequence the hypervariable regions of the mitochondrial DNA (mtDNA) genome. Non-Combined DNA Index System (CODIS) STR markers have been developed to obtain information beyond the core CODIS loci. This chapter will focus on the steps necessary to prepare and use one of the non-CODIS (NC) multiplexes, NC01 (Coble and Butler 2005), for analysis on capillary electrophoresis instrumentation. PMID:22139651

Coble, Michael D

2012-01-01

5

Development of a tetraplex PCR assay for CYP2D6 genotyping in degraded DNA samples.  

PubMed

CYP2D6 polymorphism analysis is gaining increasing interest in forensic pharmacogenetics. Nevertheless, DNA recovered from forensic samples could be of poor quality and not suitable for long polymerase chain reaction required to type CYP2D6 gene prior to SNaPshot minisequencing analysis performed to define alleles with different enzymatic activity. We developed and validated following the guidelines of the Scientific Working Group on DNA Analysis Methods a tetraplex PCR yielding four amplicons of 597, 803, 1142, and 1659 bp encompassing the entire CYP2D6 gene to analyze eleven SNP positions by SNaPshot minisequencing. Concordance, sensitivity, and specificity were assessed. The method, applied to thirty-two forensic samples failed to amplify with long PCR, allowed the amplification of CYP2D6 gene in 62.5% of degraded samples. The new tetraplex PCR appears a suitable method for CYP2D6 analysis in forensic pharmacogenetics. PMID:24313823

Riccardi, Laura N; Lanzellotto, Rossana; Falconi, Mirella; Ceccardi, Stefania; Bini, Carla; Pelotti, Susi

2014-05-01

6

qPCR and mtDNA SNP analysis of experimentally degraded hair samples and its application in forensic casework  

Microsoft Academic Search

A mitochondrial DNA (mtDNA) quantification PCR (qPCR) method was developed and applied in a study with experimentally degraded\\u000a hair samples (first study) and in a criminal case (second study). In the first study, the amount of detectable mtDNA decreased\\u000a drastically after an incubation time of 1 month on a moist tissue in a heating cabin at 37°C. In the second study,

Stephan Köhnemann; Petra Pennekamp; Peter Fritz Schmidt; Heidi Pfeiffer

2010-01-01

7

Thermal degradation of DNA.  

PubMed

In this article, we investigate the thermal degradation of deoxyribonucleic acid (DNA). We find that under dry conditions, complete DNA degradation occurs at above 190°C. In addition, as the boiling temperature of water is pressure dependent, we have investigated the thermal degradation of the DNA in water for different applied partial pressures. This information is important for fundamental understanding of DNA structure and energetics, and can be useful for biomedical applications such as thermal targeting of DNA in cancer cells, as well as for basic research. PMID:23621849

Karni, Moshe; Zidon, Dolev; Polak, Pazit; Zalevsky, Zeev; Shefi, Orit

2013-06-01

8

Sequencing Degraded DNA from Non-Destructively Sampled Museum Specimens for RAD-Tagging and Low-Coverage Shotgun Phylogenetics  

PubMed Central

Ancient and archival DNA samples are valuable resources for the study of diverse historical processes. In particular, museum specimens provide access to biotas distant in time and space, and can provide insights into ecological and evolutionary changes over time. However, archival specimens are difficult to handle; they are often fragile and irreplaceable, and typically contain only short segments of denatured DNA. Here we present a set of tools for processing such samples for state-of-the-art genetic analysis. First, we report a protocol for minimally destructive DNA extraction of insect museum specimens, which produced sequenceable DNA from all of the samples assayed. The 11 specimens analyzed had fragmented DNA, rarely exceeding 100 bp in length, and could not be amplified by conventional PCR targeting the mitochondrial cytochrome oxidase I gene. Our approach made these samples amenable to analysis with commonly used next-generation sequencing-based molecular analytic tools, including RAD-tagging and shotgun genome re-sequencing. First, we used museum ant specimens from three species, each with its own reference genome, for RAD-tag mapping. Were able to use the degraded DNA sequences, which were sequenced in full, to identify duplicate reads and filter them prior to base calling. Second, we re-sequenced six Hawaiian Drosophila species, with millions of years of divergence, but with only a single available reference genome. Despite a shallow coverage of 0.37±0.42 per base, we could recover a sufficient number of overlapping SNPs to fully resolve the species tree, which was consistent with earlier karyotypic studies, and previous molecular studies, at least in the regions of the tree that these studies could resolve. Although developed for use with degraded DNA, all of these techniques are readily applicable to more recent tissue, and are suitable for liquid handling automation.

Tin, Mandy Man-Ying; Economo, Evan Philip; Mikheyev, Alexander Sergeyevich

2014-01-01

9

The application of reduced-size short tandem repeat primer sets in the analysis of human DNA from degraded and compromised samples  

Microsoft Academic Search

The purpose of this research was to demonstrate the applicability of reduced-size STR (Miniplex) primer sets to challenging samples and to provide the forensic community with new information regarding the analysis of degraded and inhibited DNA. The Miniplex primer sets were validated in accordance with guidelines set forth by the Scientific Working Group on DNA Analysis Methods (SWGDAM) in order

Kerry Lynn Opel

2008-01-01

10

Identification of a Toluene-Degrading Bacterium from a Soil Sample through H218O DNA Stable Isotope Probing ?†  

PubMed Central

DNA stable isotope probing (DNA-SIP) with H218O was used to identify a toluene-degrading bacterium in soil amended with 48 ppm toluene. After quantification of toluene degradation rates in soil, DNA was extracted from soil incubated with H218O, H216O, H216O and 48 ppm toluene, or H218O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H216O. With extracts from soils to which only H218O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H218O and toluene was analyzed. We suggest that this third band formed because toluene does not contain any oxygen atoms and toluene-degrading organisms had to transfer oxygen atoms from H218O into metabolic intermediates to form nucleic acids de novo. We extracted the third DNA band and amplified a large fraction of the bacterial 16S rRNA gene. Direct sequencing of the PCR product obtained from the labeled DNA, as well as cloned 16S rRNA amplicons, identified a known toluene degrader, Rhodococcus jostii RHA1. A toluene-degrading bacterial strain was subsequently isolated from soil and shown to be Rhodococcus jostii RHA1. Finally, quantitative real-time PCR analysis showed that the abundance of the 16S rRNA gene of Rhodococcus jostii RHA1 increased in soil after toluene exposure but not in soils from which toluene was withheld. This study indicates that H218O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.

Woods, Angela; Watwood, Maribeth; Schwartz, Egbert

2011-01-01

11

Analysis of DNA profiles extracted from degraded samples from archival of formalin fixed tissue included in paraffin (FFTIP) and hairs  

Microsoft Academic Search

The possibility of studying DNA extracted from archival of formalin fixed tissue included in paraffin (FFTIP) enables valuable retrospective investigations. However, according to some authors it is difficult to obtain genomic DNA of good quality, since the process of fixation often results in fragmentation of DNA. In order to evaluate the quality and quantity of DNA extracted, necropsy samples of

Edna S. Miazato Iwamura; José Arnaldo Soares-Vieira; Marcelo Souza Silva; Karina S. Funabashi; Carla D. Godoy

2009-01-01

12

Resistance of degraded hair shafts to contaminant DNA  

Microsoft Academic Search

We have investigated the susceptibility of degraded human hair shaft samples to contamination by exogenous sources of DNA, including blood, saliva, skin cells, and purified DNA. The results indicate that on the whole hair shafts are either largely resistant to penetration by contaminant DNA, or extremely easy to successfully decontaminate. This pertains to samples that are both morphologically and biochemically

M Thomas P Gilbert; Laura Menez; Robert C Janaway; Desmond J Tobin; Alan Cooper; Andrew S Wilson

2006-01-01

13

Development of a SNP set for human identification: A set with high powers of discrimination which yields high genetic information from naturally degraded DNA samples in the Thai population.  

PubMed

This study describes the development of a SNP typing system for human identification in the Thai population, in particular for extremely degraded DNA samples. A highly informative SNP marker set for forensic identification was identified, and a multiplex PCR-based Invader assay was developed. Fifty-one highly informative autosomal SNP markers and three sex determination SNP markers were amplified in two multiplex PCR reactions and then detected using Invader assay reactions. The average PCR product size was 71 base pairs. The match probability of the 54-SNP marker set in 124 Thai individuals was 1.48×10(-21), higher than that of STR typing, suggesting that this 54-SNP marker set is beneficial for forensic identification in the Thai population. The selected SNP marker set was also evaluated in 90 artificially degraded samples, and in 128 naturally degraded DNA samples from real forensic casework which had shown no profiles or incomplete profiles when examined using a commercial STR typing system. A total of 56 degraded samples (44%) achieved the matching probability (PM) equivalent to STR gold standard analysis (successful genotyping of 44 SNP markers) for human identification. These data indicated that our novel 54-SNP marker set provides a very useful and valuable approach for forensic identification in the Thai population, especially in the case of highly to extremely degraded DNA. In summary, we have developed a set of 54 Thai-specific SNPs for human identification which have higher discrimination power than STR genotyping. The PCRs for these 54 SNP markers were successfully combined into two multiplex reactions and detected with an Invader assay. This novel SNP genotyping system also yields high levels of genetic information from naturally degraded samples, even though there are much more difficult to recover than artificially degraded samples. PMID:24747184

Boonyarit, Hathaichanoke; Mahasirimongkol, Surakameth; Chavalvechakul, Nuttama; Aoki, Masayuki; Amitani, Hanae; Hosono, Naoya; Kamatani, Naoyuki; Kubo, Michiaki; Lertrit, Patcharee

2014-07-01

14

Authentication of forensic DNA samples.  

PubMed

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. PMID:20129467

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

2010-02-01

15

DNA degradation of genetically modified cottonseed meal during feed processing.  

PubMed

The effects of dry heating, wet heating, and extrusion on the degradation of DNA in cottonseed meal (CSM) were studied using polymerase chain reaction (PCR) and real-time PCR approach. Both the sad1 DNA, ranging between 128 and 883 bp in size, and the cry1Ab/c gene, ranging between 183 and 652 bp in size, were detectable in all dry-heated CSM and cottonseed. During wet heating, the sad1 gene (?883 bp) and the cry1Ab/c (?952 bp) gene were thoroughly degraded at 105 and 120 °C, respectively. Sizes from 128 to 530 bp for the sad1 gene and sizes from 183 to 652 bp for the cry1Ab/c gene were detected during extrusion at temperatures ranging from 75 to 135 °C. Fragments ?883 bp for the sad1 gene and ?952 bp for the cry1Ab/c gene were detected in all of the extruded samples with water content varying between 26 and 34 %. The copy number ratio of cry1Ab/c to sad1 in samples of Bt cottonseed meal decreased rapidly when the temperature increased during the heating process. In conclusion, feed processing markedly degrades the larger DNA fragments of sad1 and cry1Ab/c, with high temperature and water content being the main factors for that degradation. PMID:23188658

Guan, Qingfeng; Wang, Xiumin; Teng, Da; Yang, Yalin; Wang, Jianhua

2013-01-01

16

Quantification of DNA in forensic samples  

Microsoft Academic Search

Quantification of DNA in a forensic sample is of major importance for proper DNA amplification and STR profiling. Several methods have been developed to quantify DNA, from basic UV spectrometry, through gel-based techniques, to dye staining, blotting techniques, and, very recently, DNA amplification methods (polymerase chain reaction, PCR). Early techniques simply measured total DNA, but newer techniques can specifically measure

Janice A. Nicklas; Eric Buel

2003-01-01

17

Forensic Strategies Used for DNA Extraction of Ancient and Degraded Museum Sturgeon Specimens  

Microsoft Academic Search

The procedure for the successful extraction of nucleic acids depends on the type of sample being extracted and the purpose\\u000a of the extraction. Protocols for the extraction of high copy number DNA samples vary significantly from those of degraded\\u000a DNA samples. This study involved the development of a DNA extraction protocol that includes a cleaning procedure designed\\u000a to remove external

E. Martinez-Espin; L. J. Martinez-Gonzalez; J. C. Alvarez; R. K. Roby; J. A. Lorente

18

Survival and DNA degradation in anhydrobiotic tardigrades.  

PubMed

Anhydrobiosis is a highly stable state of suspended animation in an organism due to its desiccation, which is followed by recovery after rehydration. Changes occurring during drying could damage molecules, including DNA. Using the anhydrobiotic tardigrade Paramacrobiotus richtersi as a model organism, we have evaluated the effects of environmental factors, such as temperature and air humidity level (RH), on the survival of desiccated animals and on the degradation of their DNA. Tardigrades naturally desiccated in leaf litter and tardigrades experimentally desiccated on blotting paper were considered. Replicates were kept at 37 degrees C and at different levels of RH for 21 days. RH values and temperature, as well as time of exposure to these environmental factors, have a negative effect on tardigrade survival and on the time required by animals to recover active life after desiccation. DNA damages (revealed as single strand breaks) occurred only in desiccated tardigrades kept for a long time at high RH values. These results indicate that during the anhydrobiotic state, damages take place and accumulate with time. Two hypotheses can be formulated to explain the results: (i) oxidative damages occur in desiccated specimens of P. richtersi, and (ii) high temperatures and high RH values change the state of the disaccharide trehalose, reducing its protective role. PMID:19946082

Rebecchi, L; Cesari, M; Altiero, T; Frigieri, A; Guidetti, R

2009-12-01

19

The effect of sample age and storage method on DNA yield and microsatellite amplification from baboon ( Papio ursinus ) faecal samples  

Microsoft Academic Search

Sampling methods that allow DNA collection without the physical handling of animals are popular in conservation genetics,\\u000a but DNA isolated from faecal samples may be degraded, potentially leading to erroneous microsatellite genotyping results.\\u000a We collected baboon faecal samples from fresh to 1-week post-defecation in a controlled sampling environment and preserved\\u000a these using three storage techniques. After DNA isolation and quantification,

Annesca Bubb; Karen Ehlers; Antoinette Kotze; J. Paul Grobler

20

Evaluating Ethanol-based Sample Preservation to Facilitate Use of DNA Barcoding in Routine Freshwater Biomonitoring Programs Using Benthic Macroinvertebrates  

EPA Science Inventory

Molecular methods, such as DNA barcoding, have the potential in enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biom...

21

Are convenience DNA samples significantly different?  

Microsoft Academic Search

In this paper, the issue of whether DNA databases collected by different convenience sampling methods are significantly different statistically is investigated. Testing the null hypothesis that the population probability or frequency distributions of DNA profiles under different sampling methods are the same is of interest in this investigation. Some statistical analyses are conducted on the single-locus VNTR databases collected from

Wing K. Fung

1996-01-01

22

DNA fingerprinting by sampled sequencing.  

PubMed Central

We describe a method for characterizing DNA segments that combines limited sequencing with size separation of restriction fragments. As part of a multistep procedure, 5' overhangs of unknown sequence are generated by cleavage with a class IIS restriction enzyme. After labeling of these ends by using dideoxynucleotides tagged with distinctive fluorescent dyes, the restriction fragments are analyzed by polyacrylamide gel electrophoresis and detection of fluorescent emissions using a commercially available DNA sequencer. The nucleotide-specific fluorescent signatures permit determination of the terminal sequence for each labeled end. The set of labeled fragments, characterized by both size and terminal sequence, constitutes a fingerprint that can be used to compare DNA segments for overlap or relatedness. The inclusion of terminal sequence data dramatically increases the information content of the fingerprint, making comparisons more reliable and efficient than those based upon size alone. Images

Brenner, S; Livak, K J

1989-01-01

23

STRs, mini STRs and SNPs--a comparative study for typing degraded DNA.  

PubMed

Short tandem repeat (STR) systems are the most powerful and widely used genetic marker systems in forensic DNA typing. Optimized amplification conditions and PCR reagents in combination with laser fluorescence based detection methods have increased the sensitivity and decreased the detection threshold down to approximately 100 pg. The quality of human DNA from forensic samples can be influenced by environmental factors. These may cause different degrees of degradation which have a negative impact on the amplification process especially of STR systems with large amplicons. Therefore, methods which need only small amplicon sizes to detect DNA markers are a better choice for typing degraded DNA. Here we report investigations on different types of DNA markers and typing methods which should all be applicable for analysing degraded DNA. These are two commercially available mini STR kits and five SNP markers which were analysed with two self established assays, a 5' nuclease assay and a minisequencing (SNaPshot) assay. The investigations comprised sensitivity studies, different types of stain material, as well as intact and degraded DNA. Results indicate that mini STRs are superior to standard STR typing methods, especially for typing old stain material with small amounts of degraded DNA. SNP typing based on the minisequencing (SNaPshot) assay achieved a better success rate in typing aged blood and saliva stains compared to standard STRs and SNP typing using the 5' nuclease assay. PMID:21269861

Senge, Tim; Madea, Burkhard; Junge, Anke; Rothschild, Markus A; Schneider, Peter M

2011-03-01

24

DNA Fingerprinting by Sampled Sequencing  

Microsoft Academic Search

We describe a method for characterizing DNA segments that combines limited sequencing with size separation of restriction fragments. As part of a multistep procedure, 5' overhangs of unknown sequence are generated by cleavage with a class IIS restriction enzyme. After labeling of these ends by using dideoxynucleotides tagged with distinctive fluorescent dyes, the restriction fragments are analyzed by polyacrylamide gel

Sydney Brenner; Kenneth J. Livak

1989-01-01

25

DNA Qualification Workflow for Next Generation Sequencing of Histopathological Samples  

PubMed Central

Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.

Simbolo, Michele; Gottardi, Marisa; Corbo, Vincenzo; Fassan, Matteo; Mafficini, Andrea; Malpeli, Giorgio; Lawlor, Rita T.; Scarpa, Aldo

2013-01-01

26

Anaerobic Methyl tert-Butyl Ether-Degrading Microorganisms Identified in Wastewater Treatment Plant Samples by Stable Isotope Probing  

PubMed Central

Anaerobic methyl tert-butyl ether (MTBE) degradation potential was investigated in samples from a range of sources. From these 22 experimental variations, only one source (from wastewater treatment plant samples) exhibited MTBE degradation. These microcosms were methanogenic and were subjected to DNA-based stable isotope probing (SIP) targeted to both bacteria and archaea to identify the putative MTBE degraders. For this purpose, DNA was extracted at two time points, subjected to ultracentrifugation, fractioning, and terminal restriction fragment length polymorphism (TRFLP). In addition, bacterial and archaeal 16S rRNA gene clone libraries were constructed. The SIP experiments indicated bacteria in the phyla Firmicutes (family Ruminococcaceae) and Alphaproteobacteria (genus Sphingopyxis) were the dominant MTBE degraders. Previous studies have suggested a role for Firmicutes in anaerobic MTBE degradation; however, the putative MTBE-degrading microorganism in the current study is a novel MTBE-degrading phylotype within this phylum. Two archaeal phylotypes (genera Methanosarcina and Methanocorpusculum) were also enriched in the heavy fractions, and these organisms may be responsible for minor amounts of MTBE degradation or for the uptake of metabolites released from the primary MTBE degraders. Currently, limited information exists on the microorganisms able to degrade MTBE under anaerobic conditions. This work represents the first application of DNA-based SIP to identify anaerobic MTBE-degrading microorganisms in laboratory microcosms and therefore provides a valuable set of data to definitively link identity with anaerobic MTBE degradation.

Sun, Weimin; Sun, Xiaoxu

2012-01-01

27

The development of miniplex primer sets for the analysis of degraded DNA  

NASA Astrophysics Data System (ADS)

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.

McCord, Bruce; Opel, Kerry; Chung, Denise; Drabek, Jiri; Tatarek, Nancy; Meadows Jantz, Lee; Butler, John

2005-05-01

28

Microfluidic DNA sample preparation method and device  

DOEpatents

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.

Krulevitch, Peter A. (Pleasanton, CA); Miles, Robin R. (Danville, CA); Wang, Xiao-Bo (San Diego, CA); Mariella, Raymond P. (Danville, CA); Gascoyne, Peter R. C. (Bellaire, TX); Balch, Joseph W. (Livermore, CA)

2002-01-01

29

SHORT REPORT: A NEW POLYMERASE CHAIN REACTIONRESTRICTION FRAGMENT LENGTH POLYMORPHISM METHOD TO IDENTIFY ANOPHELES ARABIENSIS FROM AN. GAMBIAE AND ITS TWO MOLECULAR FORMS FROM DEGRADED DNA TEMPLATES OR MUSEUM SAMPLES  

Microsoft Academic Search

We present a polymerase chain reactionrestriction fragment length polymorphism method to simulta- neously distinguish the two Anopheles gambiae M and S molecular forms and Anopheles arabiensis. This method uses different diagnostic sites than previously published methods, and it is based on the amplification of a smaller ribosomal DNA fragment. We have tested this protocol in a variety of samples from

FEDERICA SANTOLAMAZZA; ALESSANDRA DELLA TORRE; ADALGISA CACCONE

30

Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis.  

PubMed

The role of Rad51 in an unperturbed cell cycle has been difficult to distinguish from its DNA repair function. Here, using EM to visualize replication intermediates assembled in Xenopus laevis egg extract, we show that Rad51 is required to prevent the accumulation of single-stranded DNA (ssDNA) gaps at replication forks and behind them. ssDNA gaps at forks arise from extended uncoupling of leading- and lagging-strand DNA synthesis. In contrast, ssDNA gaps behind forks, which are prevalent on damaged templates, result from Mre11-dependent degradation of newly synthesized DNA strands and are suppressed by inhibition of Mre11 nuclease activity. These findings reveal direct roles for Rad51 at replication forks, demonstrating that Rad51 protects newly synthesized DNA from Mre11-dependent degradation and promotes continuous DNA synthesis. PMID:20935632

Hashimoto, Yoshitami; Ray Chaudhuri, Arnab; Lopes, Massimo; Costanzo, Vincenzo

2010-11-01

31

Degradation of chloroplast DNA during natural senescence of maple leaves.  

PubMed

The fate of chloroplast DNA (cpDNA) during plastid development and conversion between various plastid types is still not very well understood. This is especially true for the cpDNA found in plastids of naturally senescing leaves. Here, we describe changes in plastid nucleoid structure accompanied with cpDNA degradation occurring during natural senescence of the free-growing deciduous woody species Acer pseudoplatanus L. Natural senescence was investigated using three types of senescing leaves: green (G), yellow-green (YG) and yellow (Y). The extent of senescence was evaluated at the level of photosynthetic pigment degradation, accumulation of starch and plastid ultrastructure. Determination of cpDNA amount was carried out by in planta visualization with 4,6-diamidino-2-phenylindole, by Southern hybridization, and by dot-blot using an rbcL gene probe. During natural senescence, plastid nucleoids undergo structural rearrangements accompanied by an almost complete loss of cpDNA. Furthermore, senescence-associated protein components exhibiting strong binding to an ?10kbp rbcL-containg cpDNA fragment were identified. This interaction might be important for rbcL expression and Rubisco degradation during the course of natural senescence in trees. PMID:22427374

Fulgosi, Hrvoje; Jezic, Marin; Lepedus, Hrvoje; Stefanic, Petra Peharec; Curkovic-Perica, Mirna; Cesar, Vera

2012-03-01

32

Flow cytometric detection method for DNA samples  

DOEpatents

Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM.TM. on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA.TM., on the 5' end.

Nasarabadi,Shanavaz (Livermore, CA); Langlois, Richard G. (Livermore, CA); Venkateswaran, Kodumudi S. (Round Rock, TX)

2011-07-05

33

Flow cytometric detection method for DNA samples  

DOEpatents

Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM, on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA, on the 5' end.

Nasarabadi, Shanavaz (Livermore, CA); Langlois, Richard G. (Livermore, CA); Venkateswaran, Kodumudi S. (Livermore, CA)

2006-08-01

34

Molecular identification of cryptic bumblebee species from degraded samples using PCR-RFLP approach.  

PubMed

The worldwide decline and local extinctions of bumblebees have raised a need for fast and accurate tools for species identification. Morphological characters are often not sufficient, and molecular methods have been increasingly used for reliable identification of bumblebee species. Molecular methods often require high-quality DNA which makes them less suitable for analysis of low-quality or older samples. We modified the PCR-RFLP protocol for an efficient and cost-effective identification of four bumblebee species in the subgenus Bombus s. str. (B. lucorum, B. terrestris, B. magnus and B. cryptarum). We used a short partial mitochondrial COI fragment (446 bp) and three diagnostic restriction enzymes (Hinf I, Hinc II and Hae III) to identify species from degraded DNA material. This approach allowed us to efficiently determine the correct species from all degraded DNA samples, while only a subset of samples 64.6% (31 of 48) resulted in successful amplification of a longer COI fragment (1064 bp) using the previously described method. This protocol can be applied for conservation and management of bumblebees within this subgenus and is especially useful for fast species identification from degraded samples. PMID:24128053

Vesterlund, S-R; Sorvari, J; Vasemägi, A

2014-01-01

35

A general approach for DNA encapsulation in degradable polymer microcapsules.  

PubMed

We report a general and facile method for the encapsulation of DNA in nanoengineered, degradable polymer microcapsules. Single-stranded (ss), linear double-stranded (ds), and plasmid DNA were encapsulated into disulfide-cross-linked poly(methacrylic acid) (PMA) capsules. The encapsulation procedure involves four steps: adsorption of DNA onto amine-functionalized silica (SiO(2)(+)) particles; sequential deposition of thiolated PMA (PMA (SH)) and poly(vinylpyrrolidone) to form multilayers; cross-linking of the thiol groups of the PMA (SH) in the multilayers into disulfide linkages; and removal of the sacrificial SiO(2)(+) particles. Multilayer growth was dependent on the surface coverage of DNA on the SiO(2)(+) particles, with stable capsules formed from particles with up to 50% DNA surface coverage. The encapsulation strategy applies to nucleic acids with varied size and conformation and allows DNA to be concentrated over 100-fold from dilute solutions into monodisperse, uniformly loaded polymer capsules. The capsule loading can be controlled by the DNA:SiO(2)(+)particle ratio, and for 1 microm diameter capsules, loadings of approximately 1000 chains of 800 bp dsDNA and more than 10,000 chains of 20-mer ssDNA can be achieved. The encapsulated DNA was released and successfully used in polymerase chain reactions as both templates (linear dsDNA and plasmid DNA) and primer sequences (ssDNA), confirming the functionality and structural integrity of the encapsulated DNA. These DNA-loaded polymer microcapsules hold promise as delivery vehicles for gene therapy and diagnostic applications. PMID:19203131

Zelikin, Alexander N; Becker, Alisa L; Johnston, Angus P R; Wark, Kim L; Turatti, Fabio; Caruso, Frank

2007-08-01

36

DNA identification of commercial ginseng samples.  

PubMed

An investigation was performed with the objective of developing a DNA-based protocol for the identification of commercial samples of the herbal compound ginseng. There are currently two major herbal products referred to as ginseng. They are Korean or Chinese ginseng (Panax ginseng) and American ginseng (Panax quinquefolius). The market for ginseng in the United States is estimated to be approximately $300 million annually. Current tests for ginseng species identification rely on expert botanical identification of fresh plant/root specimens or on biochemical characterization of active and marker compounds (e.g., ginsenosides). For the determination of the feasibility of ginseng identification by DNA analysis, a strategy based on the direct DNA sequence analysis of the nuclear ribosomal internal transcribed spacer region was developed. Other genetic tests included sequence analysis of the chloroplast ribulose 1,5-bisphosphate carboxylase large subunit gene and DNA fingerprinting by the rapid amplification of polymorphic DNA technique. To confirm the results, each ginseng sample was identified using high-performance liquid chromatography. All methods were successful in distinguishing American from Korean ginseng. In addition, the protocol was improved for the isolation of genomic and plastid DNA from commercial ginseng preparations by incorporating an impact homogenization step into the standard column chromatography purification procedure. PMID:10956181

Mihalov, J J; Marderosian, A D; Pierce, J C

2000-08-01

37

Adsorption of DNA to sand and variable degradation rates of adsorbed DNA.  

PubMed Central

Adsorption and desorption of DNA and degradation of adsorbed DNA by DNase I were studied by using a flowthrough system of sand-filled glass columns. Maximum adsorption at 23 degrees C occurred within 2 h. The amounts of DNA which adsorbed to sand increased with the salt concentration (0.1 to 4 M NaCl and 1 mM to 0.2 M MgCl2), salt valency (Na+ less than Mg2+ and Ca2+), and pH (5 to 9). Maximum desorption of DNA from sand (43 to 59%) was achieved when columns were eluted with NaPO4 and NaCl for 6 h or with EDTA for 1 h. DNA did not desorb in the presence of detergents. It is concluded that adsorption proceeded by physical and chemical (Mg2+ bridging) interaction between the DNA and sand surfaces. Degradability by DNase I decreased upon adsorption of transforming DNA. When DNA adsorbed in the presence of 50 mM MgCl2, the degradation rate was higher than when it adsorbed in the presence of 20 mM MgCl2. The sensitivity to degradation of DNA adsorbed to sand at 50 mM MgCl2 decreased when the columns were eluted with 0.1 mM MgCl2 or 100 mM EDTA before application of DNase I. This indicates that at least two types of DNA-sand complexes with different accessibilities of adsorbed DNA to DNase I existed. The degradability of DNA adsorbed to minor mineral fractions (feldspar and heavy minerals) of the sand differed from that of quartz-adsorbed DNA.

Lorenz, M G; Wackernagel, W

1987-01-01

38

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2010 CFR

...2010-07-01 2010-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing §...

2010-07-01

39

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2010 CFR

...ADMINISTRATION] [Chapter I - DEPARTMENT OF JUSTICE] [Part 28 - DNA IDENTIFICATION SYSTEM] [Subpart B - DNA Sample Collection, Analysis, and Indexing] [Sec. 28.12 - Collection of DNA samples.] 28 JUDICIAL ADMINISTRATION 1...

2009-07-01

40

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2013 CFR

...2013-07-01 2013-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing §...

2013-07-01

41

Metagenomics: DNA sequencing of environmental samples  

SciTech Connect

While genomics has classically focused on pure,easy-to-obtain samples, such as microbes that grow readily in culture orlarge animals and plants, these organisms represent but a fraction of theliving or once living organisms of interest. Many species are difficultto study in isolation, because they fail to grow in laboratory culture,depend on other organisms for critical processes, or have become extinct.DNA sequence-based methods circumvent these obstacles, as DNA can bedirectly isolated from live or dead cells in a variety of contexts, andhave led to the emergence of a new field referred to asmetagenomics.

Tringe, Susannah Green; Rubin, Edward M.

2005-09-01

42

Mitochondrial DNA analysis of Swedish population samples.  

PubMed

As a contribution to the geographic coverage of EMPOP, currently the best available forensic mitochondrial DNA (mtDNA) database, a total of 299 Swedish individuals were analysed by sequencing of the first and second hypervariable regions of the mtDNA genome. In this sample set, a total of 179 different haplotypes were detected. The genetic diversity was estimated to be 0.9895 (±0.0023), and the random match probability was 1.39 %. The most abundant haplogroups were HV (including its subhaplogroups H andV) with a frequency of 46.5%, followed by haplogroup U(including its subhaplogroup K) at 27.8 %, haplogroup T at 10.0 % and haplogroup J at 7.0 %, a distribution that is consistent with previous observations in other European populations. PMID:24077990

Lembring, Maria; van Oven, Mannis; Montelius, Maria; Allen, Marie

2013-11-01

43

Comparative analysis of human mitochondrial DNA from World War I bone samples by DNA sequencing and ESI-TOF mass spectrometry  

Microsoft Academic Search

Mitochondrial DNA is commonly used in identity testing for the analysis of old or degraded samples or to give evidence of familial links. The Abbott T5000 mass spectrometry platform provides an alternative to the more commonly used Sanger sequencing for the analysis of human mitochondrial DNA. The robustness of the T5000 system has previously been demonstrated using DNA extracted from

Rebecca Howard; Vesela Encheva; Jim Thomson; Katherine Bache; Yuen-Ting Chan; Simon Cowen; Paul Debenham; Alan Dixon; Jens-Uwe Krause; Elaina Krishan; Daniel Moore; Victoria Moore; Michael Ojo; Sid Rodrigues; Peter Stokes; James Walker; Wolfgang Zimmermann; Rita Barallon

44

Degradable starch nanoparticle assisted ethanol precipitation of DNA.  

PubMed

Precipitation of DNA from a large volume of aqueous solution is an important step in many molecular biology and analytical chemistry experiments. Currently, this is mainly achieved by ethanol precipitation, where a long-term incubation (usually overnight) at low temperature of -20 to -80°C with high salt concentration is required. This method also requires a large quantity of DNA to form a visible pellet and was tested mainly for double-stranded DNA. To improve DNA precipitation, co-precipitating polymers such as linear polyacrylamide has been used. In this work, we report that starch nanoparticles (SNPs) can achieve convenient DNA precipitation at room temperature with a low salt concentration and short incubation time. This method requires as low as 0.01-0.1% SNPs and can precipitate both single- and double-stranded DNA of various lengths. The effect of salt concentration, pH and the crosslinking density of SNPs has been systematically studied. Compared to other types of precipitating agents, SNPs are highly biocompatible and can be degraded by a common enzyme (amylase). This work suggests a novel application of a bio-based material that is prepared in mass production. PMID:24906766

Ip, Alexander C-F; Tsai, Tsung Hao; Khimji, Imran; Huang, Po-Jung Jimmy; Liu, Juewen

2014-09-22

45

Characterization of New MiniSTR Loci to Aid Analysis of Degraded DNA  

Microsoft Academic Search

ABSTRACT: AnumberofstudieshavedemonstratedthatsuccessfulanalysisofdegradedDNAspecimensfrommassdisastersorforensicevidence improves with smaller sized polymerase chain reaction (PCR) products. We have scanned the literature for new STR loci, unlinked from the CODIS markers, which can generate amplicons less than 125bp in size and would therefore be helpful in testing degraded DNA samples. New PCR primers were designed and tested for the STR loci D1S1677, D2S441, D4S2364, D10S1248, D14S1434,

Michael D. Coble; John M. Butler

2005-01-01

46

Automatable full demineralization DNA extraction procedure from degraded skeletal remains.  

PubMed

During the 7 year period from 2002 to 2009 a high volume, silica-binding DNA extraction protocol for bone, based on modified QIAGEN's Blood Maxi Kit protocol was highly successful permitting the DNA matching of >14,500 missing persons from former Yugoslavia. This method, however, requires large amount of bone material and large volumes of reagents. The logical evolution was to develop a more efficient extraction protocol for bone samples that uses significantly less starting material while increasing the success in obtaining DNA results from smaller, more challenging samples. In this study we compared the performance of ICMP's original protocol against an automatable full demineralization approach. In order to provide reliable results and to simulate a wide variety of cases, we analyzed 40 bone samples in a comparative study based on DNA concentrations and quality of resulting STR profiles. The new protocol results in the dissolution of the entire bone powder sample, thus eliminating the possibility that DNA is left behind, locked in remaining solid bone matrix. For the majority of samples tested, the DNA concentrations obtained from half a gram of fully digested bone material were equivalent to or greater than the ones obtained from 2g of partially demineralized bone powder. Furthermore, the full demineralization process significantly increases the proportion of full profiles reflecting the correlation with better DNA quality. This method has been adapted for the QIAcube robotic platform. The performance of this automated full demineralization protocol is similar to the manual version and increases overall lab throughput. It also simplifies the process by eliminating quality control procedures that are advisable in manual procedures, and overall reduces the chance of human error. Finally we described a simple and efficient post-extraction clean-up method that can be applied to DNA extracts obtained from different protocols. This protocol has also been adjusted for the QIAcube platform. PMID:21885362

Amory, Sylvain; Huel, René; Bili?, Ana; Loreille, Odile; Parsons, Thomas J

2012-05-01

47

Characterizing DNA preservation in degraded specimens of Amara alpina (Carabidae: Coleoptera).  

PubMed

DNA preserved in degraded beetle (Coleoptera) specimens, including those derived from dry-stored museum and ancient permafrost-preserved environments, could provide a valuable resource for researchers interested in species and population histories over timescales from decades to millenia. However, the potential of these samples as genetic resources is currently unassessed. Here, using Sanger and Illumina shotgun sequence data, we explored DNA preservation in specimens of the ground beetle Amara alpina, from both museum and ancient environments. Nearly all museum specimens had amplifiable DNA, with the maximum amplifiable fragment length decreasing with age. Amplification of DNA was only possible in 45% of ancient specimens. Preserved mitochondrial DNA fragments were significantly longer than those of nuclear DNA in both museum and ancient specimens. Metagenomic characterization of extracted DNA demonstrated that parasite-derived sequences, including Wolbachia and Spiroplasma, are recoverable from museum beetle specimens. Ancient DNA extracts contained beetle DNA in amounts comparable to museum specimens. Overall, our data demonstrate that there is great potential for both museum and ancient specimens of beetles in future genetic studies, and we see no reason why this would not be the case for other orders of insect. PMID:24266987

Heintzman, Peter D; Elias, Scott A; Moore, Karen; Paszkiewicz, Konrad; Barnes, Ian

2014-05-01

48

Multiple DNA Extractions Coupled with Stable-Isotope Probing of Anthracene-Degrading Bacteria in Contaminated Soil?†  

PubMed Central

In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the 13C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-13C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from 13C-enriched DNA and were designated “anthracene group 1.” Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP.

Jones, Maiysha D.; Singleton, David R.; Sun, Wei; Aitken, Michael D.

2011-01-01

49

Forensic Mitochondrial DNA Analysis of 116 Casework Skeletal Samples  

Microsoft Academic Search

Between February 1999 and May 2005, 116 DNA extractions were completed on skeletal remains from routine casework. Overall, at least a partial mitochondrial DNA (mtDNA) profile was obtained on 83.6% of samples. Skeletal remains fell into two general categories: (1) samples for body identifications submitted by law enforcement and (2) samples submitted to answer historical or family identity questions. Body

Kimberlyn Nelson; Terry Melton

2007-01-01

50

Degradation of transgene DNA in genetically modified herbicide-tolerant rice during food processing  

Microsoft Academic Search

In order to assess the effect of food processing on the degradation of exogenous DNA components in sweet rice wine and rice crackers made from genetically modified (GM) rice (Oryza sativa L.), we developed genomic DNA extraction methods and compared the effect of different food processing procedures on DNA degradation. It was found that the purity, quantity and quality of

Shangxin Song; Guanghong Zhou; Feng Gao; Wei Zhang; Liangyan Qiu; Sifa Dai; Xinglian Xu; Hongmei Xiao

2011-01-01

51

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

PubMed Central

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.

Iversen, Lars L?nsmann; M?ller, Peter Rask; Rasmussen, Morten; Willerslev, Eske

2012-01-01

52

Forensic DNA typing strategy of degraded DNA on discarded cigarette ends using the AmpF?STR® Identifiler®, Identifiler® Plus and MiniFiler™ PCR Amplification Kits.  

PubMed

DNA left on a forensic sample is often prone to degradation, especially if left to the elements. To maximize the chance of retrieving the most information from such compromised DNA, an appropriate profiling scheme using the available technologies needs to be devised. In this study, a total of 62 cigarette ends collected under different conditions of environmental exposure were employed to test the effectiveness of three DNA amplification kits, namely the Applied Biosystems™ AmpF?STR® Identifiler®, Identifiler® Plus and MiniFiler™ PCR Amplification Kits, in the profiling of such compromised DNA. We demonstrated that Identifiler® Plus could substitute Identifiler® to improve the effectiveness of profiling for those inhibited cigarette samples. MiniFiler™, on the other hand, could supplement Identifiler®/Identifiler® Plus profiles and provide additional genetic information to enhance the evidential value of the samples, especially for those that have suffered from DNA degradation to a greater extent. The findings in this work allowed us to propose a DNA profiling strategy as follow: 1) samples yielding complete Identifiler®/Identifiler® Plus profiles require no further testing with MiniFiler™; 2) samples yielding partial single-source profiles to be tested with MiniFiler™ to add genetic information; 3) samples yielding no results are unlikely to yield any results with MiniFiler™. PMID:25002050

Ip, Stephen C Y; Lin, Sze-Wah; Li, Christina; Lai, Kam-Ming

2014-07-01

53

Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review  

Microsoft Academic Search

The applicability of a DNA-based method for GMO detection and quantification depends on the quality and quantity of the DNA.\\u000a Important food-processing conditions, for example temperature and pH, may lead to degradation of the DNA, rendering PCR analysis\\u000a impossible or GMO quantification unreliable. This review discusses the effect of several food processes on DNA degradation\\u000a and subsequent GMO detection and

Nicolas Gryson

2010-01-01

54

Proteasomal degradation of herpes simplex virus capsids in macrophages releases DNA to the cytosol for recognition by DNA sensors1  

PubMed Central

The innate immune system is important for control of infections, including herpesvirus infections. Intracellular DNA potently stimulates antiviral IFN responses. It is known that plasmacytoid dendritic cells sense herpesvirus DNA in endosomes via TLR9, and that non-immune tissue cells can sense herpesvirus DNA in the nucleus. However, it remains unknown how and where myeloid cells, like macrophages and conventional dendritic cells, detect infections with herpesviruses. Here we demonstrate that the HSV-1 capsid was ubiquitinated in the cytosol and degraded by the proteasome, hence releasing genomic DNA into the cytoplasm for detection by DNA sensors. In this context, the DNA sensor IFI16 is important for induction of IFN-? in human macrophages after infection with HSV-1 and CMV. Viral DNA localized to the same cytoplasmic regions as IFI16, with DNA sensing being independent of viral nuclear entry. Thus, proteasomal degradation of herpesvirus capsids releases DNA to the cytoplasm for recognition by DNA sensors.

Horan, Kristy A.; Hansen, Kathrine; Jakobsen, Martin R.; Holm, Christian K.; S?by, Stine; Unterholzner, Leonie; Thompson, Mikayla; West, John A.; Iversen, Marie B.; Rasmussen, Simon B.; Ellermann-Eriksen, Svend; Kurt-Jones, Evelyn; Landolfo, Santo; Damania, Blossom; Melchjorsen, Jesper; Bowie, Andrew G.; Fitzgerald, Katherine A.; Paludan, S?ren R.

2013-01-01

55

Electrochemical DNA biosensor for analysis of wastewater samples  

Microsoft Academic Search

The application of a disposable electrochemical DNA biosensor to wastewater samples is reported. The DNA biosensor is assembled by immobilising double-stranded calf thymus DNA on the surface of a disposable, carbon screen-printed electrode (SPE). The oxidation signal of the guanine base, obtained by a square wave voltammetric scan, is used as analytical signal. The presence of compounds with affinity for

F Lucarelli; A Kicela; I Palchetti; G Marrazza; M Mascini

2002-01-01

56

A DNA binding motif coordinating synthesis and degradation in proofreading DNA polymerases.  

PubMed Central

The functional significance of the conserved motif 'YxGG/A', located between the 3'-5' exonuclease and polymerization domains of eukaryotic-type DNA polymerases, has been studied by site-directed mutagenesis in phi29 DNA polymerase. Single substitutions at this region were obtained, and 11 phi29 DNA polymerase mutant derivatives were overproduced in Escherichia coli and purified to homogeneity. Nine mutants showed an altered polymerase/3'-5' exonuclease balance on a template/primer DNA structure, giving rise to three different mutant phenotypes: (i) favored polymerization (high pol/exo ratio); (ii) favored exonucleolysis (low pol/exo ratio); and (iii) favored exonucleolysis and null polymerization. Interestingly, these three different phenotypes could be obtained by mutating a single amino acid at the 'YxGG/A' motif. All different phenotypes could be directly related to defects in DNA binding at a particular active site. Thus, a high pol/exo ratio was related to a poor stability at the 3'-5' exonuclease active site. On the contrary, a low pol/exo ratio or null polymerization capacity was related to a poor stability at the polymerization active site and either a normal or an increased accessibility to the exonuclease active site. These results allow us to propose that this motif, located in the connecting region between the N-terminal and C-terminal domains, has a primary role in DNA binding, playing a critical role in the coordination or cross-talk between synthesis and degradation. Images

Truniger, V; Lazaro, J M; Salas, M; Blanco, L

1996-01-01

57

Y-STR analysis of degraded DNA using reduced-size amplicons.  

PubMed

To increase the success rate of Y-STR genotyping for degraded DNA, we have developed two multiplex PCR sets for 21 Y-STR loci. Besides the 17 Y-STR loci of DYS19, DYS385, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, and GATA H4.1 contained in a commercial Y-STR kit, AmpFlSTR Yfiler, the other four loci of DYS388, DYS446, DYS447, and DYS449 were also included in the multiplexes to increase the discrimination capacity. Among a total of 21 Y-STR loci, the primers for eight loci (DYS385, DYS390, DYS438, DYS446, DYS448, DYS449, and DYS635) were newly designed in the present study and nine loci (DYS385, DYS390, DYS391, DYS392, DYS438, DYS439, DYS448, and DYS635) have PCR amplicons smaller than those of the AmpFlSTR Yfiler kit. A sensitivity test using serially diluted standard 9948 male DNA showed that all the values of Y-STR loci in the Y-miniplexes are reliable at template concentrations as low as 30 pg. We compared the effectiveness of the two multiplexes with the AmpFlSTR Yfiler kit by using both enzymatically degraded DNA and 30 samples of 50-year-old skeletal remains. This comparison demonstrated that the new Y-miniplex sets can produce a better signal from degraded DNA than the AmpFlSTR Yfiler kit. PMID:17106735

Park, Myung Jin; Lee, Hwan Young; Chung, Ukhee; Kang, Seung-Chul; Shin, Kyoung-Jin

2007-03-01

58

A biomechatronic fluid-sample-handling system for DNA processing  

Microsoft Academic Search

An automated biomechatronic small fluid sample preparation system for deoxyribonucleic acid (DNA) processing has been developed in the Genomation Laboratory of the Department of Electrical Engineering, University of Washington, Seattle, WA. The system automates steps necessary to prepare DNA for sequencing, using sample volumes ten times smaller than the current state-of-the-art manual and automated instrumentation. This will significantly reduce the

Deirdre R. Meldrum

1997-01-01

59

The extraction of fungal DNA from multiple large soil samples  

Microsoft Academic Search

A commercial electric paint shaker was utilized to enable the simultaneous extraction of DNA from multiple large soil samples. A bead homogenization procedure was used to extract DNA from soil samples added to tubes. Soils infested with chlamydospores of the phytopathogenic fungus Cylindrocarpon destructans were used to evaluate the procedure. Glass beads and zirconium-oxide beads of various diameters were compared

R. D. Reeleder; B. B. Capell; L. D. Tomlinson; W. J. Hickey

2003-01-01

60

Long-term exposure to a pulsed magnetic field (1.5?mT, 25?Hz) increases genomic DNA spontaneous degradation.  

PubMed

Abstract The aim of this work is to investigate whether long-term pulsed magnetic field (MF) has genotoxic activity by induction of DNA damage on DNA molecules in vitro, in the absence of repair mechanisms. Yeast genomic DNA prepared by phenol extraction from S. cerevisiae cultures and the commercial DNA molecular weight marker Hyperladder I (HL-I) were exposed to 1.5?mT peak, pulsed 25?Hz MF, 8?h/day, 16 days. The total content of DNA (undamaged and damaged DNA) decreased during the exposure of genomic DNA to MF. On day 16 of exposure the DNA content was 41?±?8.1%. In addition, the undamaged DNA decreases until 6.2?±?3.1% for unexposed control samples and until 0.3?±?0.1% for pulsed MF-treated samples at day 16 of exposure. Therefore, the pulsed MF induced at day 16 an increase of 20.7-fold more degradation of DNA molecules >10?000?bp (undamaged DNA) than that observed for unexposed control samples. However, no effect was observed for HL-I DNA marker exposures. We conclude that long-term exposure to a pulsed MF (1.5?mT peak, 25?Hz, 8?h/day, 16 days) induces an increment in the DNA spontaneous degradation of yeast genomic DNA. PMID:23781973

López-Díaz, Beatriz; Mercado-Sáenz, Silvia; Martínez-Morillo, Manuel; Sendra-Portero, Francisco; Ruiz-Gómez, Miguel J

2014-09-01

61

Membrane-mediated sample loading for automated DNA sequencing.  

PubMed

A new and improved sample loading method for DNA sequencing gels using oligo/polynucleotide binding membranes is described. The labeled DNA sequencing fragments were spotted onto a membrane sample loader, which was then placed in contact with the precast polyacrylamide separation gel. In this way, the time-consuming sample well loading by the tedious pipetting procedure was avoided. The spotted DNA fragments remain immobilized on the membrane until the separation process is initiated by the application of the electric field (an active DNA release mechanism). This novel technique enables sample loading outside of the separation platform, thereby allowing full utilization of various automated sample preparation and liquid handling (robotics) systems, resulting in "real" automated DNA sequencing. The loaded membranes can be stored for more than 24 hours for later use. PMID:9694278

Cassel, S M; Guttman, A

1998-06-01

62

A new sensitive short pentaplex (ShoP) PCR for typing of degraded DNA  

Microsoft Academic Search

Analysis of short tandem repeat makers has become the most powerful tool for DNA typing in forensic casework analysis. Unfortunately, typing of DNA extracted from telogen shed hairs, bones buried in the soil or from paraffin-embedded, formalin-fixed tissue often reveals no results due to the degradation of DNA. The reduction in size of the target fragments by development of new

C. Meissner; P. Bruse; E. Mueller; M. Oehmichen

2007-01-01

63

Magnetic scanometric DNA microarray detection of methyl tertiary butyl ether degrading bacteria for environmental monitoring  

Microsoft Academic Search

A magnetoresistive biosensing platform based on a single magnetic tunnel junction (MTJ) scanning probe and DNA microarrays labeled with magnetic particles has been developed to provide an inexpensive, sensitive and reliable detection of DNA. The biosensing platform was demonstrated on a DNA microarray assay for quantifying bacteria capable of degrading methyl tertiary butyl ether (MTBE), where concentrations as low as

Mei-Lin Chan; Gerardo Jaramillo; Krassimira R. Hristova; David A. Horsley

2011-01-01

64

Circulating cell-free DNA levels increase variably following chorionic villus sampling  

PubMed Central

Objective Cell-free fetal DNA (cffDNA) in maternal plasma results from degradation of fetal and/or placental cells. Our objective was to determine if chorionic villus sampling (CVS) causes increased release of fetal and/or maternal DNA. Methods Fifty-two pregnant women were recruited prior to CVS, performed for clinical indications, at 10 5/7 to 13 2/7 weeks. Maternal blood was collected before and within 15 minutes after CVS. cffDNA was extracted from plasma. Real-time polymerase chain reaction (PCR) amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the Y chromosome sequence DYS1 were used as measures of total and fetal DNA, respectively. All samples were analyzed in triplicate without knowledge of fetal gender. Results Sensitivity of DYS1 detection in male fetuses was 100% (n=30); specificity in female fetuses was 100% (n=22). While a majority of women had >50% post-procedure increases in both fetal and total DNA, some showed post-procedure decreases. However, overall median proportional increases were not statistically significant. Gestational age (GA), placental location, and individual CVS operator did not correlate with changes in DNA levels. Conclusions While there were no statistically significant overall changes in DNA levels after CVS, as-yet undiscovered variables may influence the extent of post-procedure release of cell-free DNA in the circulation of pregnant women.

Vora, Neeta L.; Johnson, Kirby L.; Peter, Inga; Tighiouart, Hocine; Ralston, Steven J.; Craigo, Sabrina D.; Bianchi, Diana W.

2010-01-01

65

NUC-1, a Caenorhabditis elegans DNase II homolog, functions in an intermediate step of DNA degradation during apoptosis  

Microsoft Academic Search

One hallmark of apoptosis is the degradation of chromosomal DNA. We cloned the Caenorhabditis elegans gene nuc-1, which is involved in the degradation of the DNA of apoptotic cells, and found that nuc-1 encodes a homolog of mammalian DNase II. We used the TUNEL technique to assay DNA degradation in nuc-1 and other mutants defective in programmed cell death and

Yi-Chun Wu; Gillian M. Stanfield; H. Robert Horvitz

2000-01-01

66

Pooling hair samples to increase DNA yield for PCR  

Microsoft Academic Search

Hairs are useful non-invasive sources of DNA, but the DNA yield can be very small, thus promoting genotyping errors. Using\\u000a multiple hairs can counter this problem, but may introduce multiple contributors to a sample if collected remotely. With microsatellite\\u000a genotyping, samples representing multiple animals are obvious if three or more alleles are detected at any locus: these samples\\u000a can then

D. L. Alpers; A. C. Taylor; P. Sunnucks; S. A. Bellman; W. B. Sherwin

2003-01-01

67

DNA identification of Salvia divinorum samples.  

PubMed

Salvia divinorum (diviner's sage) is a plant in the mint family that produces an hallucinogenic compound, salvinorin A. The plant is used, often by chewing or smoking, as a "recreational" drug source and is regulated or banned in several states and countries. We describe a simple DNA technique, polymerase chain reaction of the ribulose bisphosphate carboxylase large subunit (rbcL) gene, that can distinguish S. divinorum leaf pieces from pieces of tobacco or cannabis. We have also found DNA sequences adjacent to the chloroplast leucine transfer RNA (trnL) gene that are specific to S. divinorum and distinguish it from other horticulturally popular Salvia species. We report some significant differences between the S. divinorum trnL sequences we determined and those now published in GenBank. PMID:22578875

Murphy, Terence M; Bola, Gurpreet

2013-01-01

68

Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review.  

PubMed

The applicability of a DNA-based method for GMO detection and quantification depends on the quality and quantity of the DNA. Important food-processing conditions, for example temperature and pH, may lead to degradation of the DNA, rendering PCR analysis impossible or GMO quantification unreliable. This review discusses the effect of several food processes on DNA degradation and subsequent GMO detection and quantification. The data show that, although many of these processes do indeed lead to the fragmentation of DNA, amplification of the DNA may still be possible. Length and composition of the amplicon may, however, affect the result, as also may the method of extraction used. Also, many techniques are used to describe the behaviour of DNA in food processing, which occasionally makes it difficult to compare research results. Further research should be aimed at defining ingredients in terms of their DNA quality and PCR amplification ability, and elaboration of matrix-specific certified reference materials. PMID:20012944

Gryson, Nicolas

2010-03-01

69

Improved Detection of Male DNA in Post-Coital Samples.  

National Technical Information Service (NTIS)

Male DNA-containing samples collected from sexual assault or homicide victims can contain very low levels of cellular male DNA admixed with a large number of female epithelial cells. This often results in failure to obtain an autosomal STR profile from th...

A. van Daal H. Lubenow J. Ballantyne

2012-01-01

70

Microfluidic devices for DNA sequencing: sample preparation and electrophoretic analysis  

Microsoft Academic Search

Modern DNA sequencing ‘factories’ have revolutionized biology by completing the human genome sequence, but in the race to completion we are left with inefficient, cumbersome, and costly macroscale processes and supporting facilities. During the same period, microfabricated DNA sequencing, sample processing and analysis devices have advanced rapidly toward the goal of a ‘sequencing lab-on-a-chip’. Integrated microfluidic processing dramatically reduces analysis

Brian M Paegel; Robert G Blazej; Richard A Mathies

2003-01-01

71

Current developments in forensic interpretation of mixed DNA samples (Review)  

PubMed Central

A number of recent improvements have provided contemporary forensic investigations with a variety of tools to improve the analysis of mixed DNA samples in criminal investigations, producing notable improvements in the analysis of complex trace samples in cases of sexual assult and homicide. Mixed DNA contains DNA from two or more contributors, compounding DNA analysis by combining DNA from one or more major contributors with small amounts of DNA from potentially numerous minor contributors. These samples are characterized by a high probability of drop-out or drop-in combined with elevated stutter, significantly increasing analysis complexity. At some loci, minor contributor alleles may be completely obscured due to amplification bias or over-amplification, creating the illusion of additional contributors. Thus, estimating the number of contributors and separating contributor genotypes at a given locus is significantly more difficult in mixed DNA samples, requiring the application of specialized protocols that have only recently been widely commercialized and standardized. Over the last decade, the accuracy and repeatability of mixed DNA analyses available to conventional forensic laboratories has greatly advanced in terms of laboratory technology, mathematical models and biostatistical software, generating more accurate, rapid and readily available data for legal proceedings and criminal cases.

HU, NA; CONG, BIN; LI, SHUJIN; MA, CHUNLING; FU, LIHONG; ZHANG, XIAOJING

2014-01-01

72

XPB mediated retroviral cDNA degradation coincides with entry to the nucleus  

SciTech Connect

Retroviruses must integrate their cDNA to a host chromosome, but a significant fraction of retroviral cDNA is degraded before integration. XPB and XPD are part of the TFIIH complex which mediates basal transcription and DNA nucleotide excision repair. Retroviral infection increases when XPB or XPD are mutant. Here we show that inhibition of mRNA or protein synthesis does not affect HIV cDNA accumulation suggesting that TFIIH transcription activity is not required for degradation. Other host factors implicated in the stability of cDNA are not components of the XPB and XPD degradation pathway. Although an increase of retroviral cDNA in XPB or XPD mutant cells correlates with an increase of integrated provirus, the integration efficiency of pre-integration complexes is unaffected. Finally, HIV and MMLV cDNA degradation appears to coincide with nuclear import. These results suggest that TFIIH mediated cDNA degradation is a nuclear host defense against retroviral infection.

Yoder, Kristine E., E-mail: yoder.176@osu.ed [Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University Medical Center and Comprehensive Cancer Center (United States) and Human Cancer Genetics, Ohio State University Medical Center and Comprehensive Cancer Center (United States); Roddick, William; Hoellerbauer, Pia [Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University Medical Center and Comprehensive Cancer Center (United States); Fishel, Richard, E-mail: rfishel@osu.ed [Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University Medical Center and Comprehensive Cancer Center (United States); Human Cancer Genetics, Ohio State University Medical Center and Comprehensive Cancer Center (United States); Physics Department, Ohio State University Columbus, OH 43210 (United States)

2011-02-20

73

Miniaturized DNA Biosensor Systems for Detecting 'Cryptosporidium' in Water Samples.  

National Technical Information Service (NTIS)

This project led to the development of novel biosensing protocols for detecting Cryptosporidium in water samples. The effort focused on new miniaturized biosensors, that convert the DNA recognition (hybridization) process into electrical or frequency sign...

J. Wang

2000-01-01

74

Integrated Systems for DNA Sample Preparation and Detection in Environmental Samples  

SciTech Connect

Field-portable sensor systems are currently needed for the detection and characterization of biological pathogens in the enviornment. Nucleic acid analysis is frequently the method of choice for discriminating between pathogenic and non-pathogenic bacteria in environmental samples, however standard protocls are difficult to automate and current microfluidic devices are not configured to analyze environment samples. In this paper, we describe an automated DNA sample processing system and demostrate its use for the extraction of bacterial DNA from water and sediment samples. Two challenges in environmental sample analysis are the need to process relatively large sample volumes (microliters to many milliliters) in order to obtain detectable quantities of DNA present at low concenntrations, and the need to purify DNA from a complex sample matrix for downstream detection. These problems are addressed by using sequential injection fluid handling techniques for precise manipulation of the required volumes, and renewable separation columns for automatically trapping and releasing microparticles that are used for sample purification. The renewable microcolumns are used for both bacteria cell concentration and DNA purification. The purified bacteria DNA is then amplified using an on-line PCR module in order to produce detectable quantities of the target DNA.

Bruckner-Lea, Cindy J.; Anheier, Norman C.; Holman, David A.; Tsukuda, Toyoko; Kingsley, Mark T.; Brockman, Fred J.; Price, Jane M.; Grate, Jay W.; Chandler, Darrell P.

2000-06-01

75

Evaluation of purification procedures of DNA from maize-meal samples by exploiting different analytical techniques for the assessment of DNA quality.  

PubMed

Two different approaches generally applied to achieve purification of DNA extracted from cells were compared: precipitation by organic solvents and enzymatic treatments. We investigated various experimental protocols reported in literature by evaluating DNA purity, integrity and yield. Reliability of analytical techniques normally employed to assess DNA purity and quantity was studied and comments and conclusions were suggested by comparing results obtained by different analytical techniques. Enzymatic treatments prove to be unable of increasing DNA purity while determining a significant degradation. In contrast, optimised conditions for solvent precipitation enabled a sharp increase of DNA purity to be obtained, associated with the maintenance of the initial DNA integrity. The application of the optimised protocol to maize-meal samples allowed us to achieve a good PCR amplification even with those samples which gave poor amplification by following the protocol recommended by the Italian legislation in force for GMO detection in food. PMID:15242092

Vanni, Adriano; Anfossi, Laura; Giovannoli, Cristina; Oddenino, Leila; Giraudi, Gianfranco

2004-04-01

76

Preparation of DNA and nucleoprotein samples for AFM imaging  

PubMed Central

Sample preparation techniques allowing reliable and reproducible imaging of DNA with various structures, topologies and complexes with proteins are reviewed. The major emphasis is given to methods utilizing chemical functionalization of mica, enabling preparation of the surfaces with required characteristics. The methods are illustrated by examples of imaging of different DNA structures. Special attention is given to the possibility of AFM to image the dynamics of DNA at the nanoscale. The capabilities of time-lapse AFM in aqueous solutions are illustrated by imaging of dynamic processes as transitions of local alternative structures (transition of DNA between H and B forms). The application of AFM to studies of protein-DNA complexes is illustrated by a few examples of imaging site-specific complexes, as well as such systems as chromatin. The time-lapse AFM studies of protein-DNA complexes including very recent advances with the use of high-speed AFM are reviewed.

Lyubchenko, Yuri L.

2010-01-01

77

RNA cell typing and DNA profiling of mixed samples: can cell types and donors be associated?  

PubMed

Forensic samples regularly involve mixtures, which are readily recognised in forensic analyses. Combined DNA and mRNA profiling is an upcoming forensic practice to examine donors and cell types from the exact same sample. From DNA profiles individual genotypes may be deconvoluted, but to date no studies have established whether the cell types identified in corresponding RNA profiles can be associated with individual donors. Although RNA expression levels hold many variables from which an association may not be expected, proof of concept is important to forensic experts who may be cross examined about this possible correlation in court settings. Clearly, the gender-specificity of certain body fluids (semen, vaginal mucosa, menstrual secretion) can be instructive. However, when donors of the same gender or gender-neutral cell types are involved, alternatives are needed. Here we analyse basic two-component mixtures (two cell types provided by different donors) composed of six different cell types, and assess whether the heights of DNA and RNA peaks may guide association of donor and cell type. Divergent results were obtained; for some mixtures RNA peak heights followed the DNA results, but for others the major DNA component did not present higher RNA peaks. Also, variation in mixture ratios was observed for RNA profiling replicates and when different donor couples gave the same two body fluids. As sample degradation may affect the two nucleic acids and/or distinct cell types differently (and thus influence donor and cell type association), mixtures were subjected to elevated temperature or UV-light. Variation in DNA and RNA stability was observed both between and within cell types and depended on the method inducing degradation. Taken together, we discourage to associate cell types and donors from peak heights when performing RNA and DNA profiling. PMID:23937933

Harteveld, Joyce; Lindenbergh, Alexander; Sijen, Titia

2013-09-01

78

The Application of Miniplex Primer Sets in the Analysis of Degraded DNA from Human Skeletal Remains  

Microsoft Academic Search

A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA

Kerry L. Opel; Denise T. Chung; Jiri Drabek; Nancy E. Tatarek; Lee Meadows Jantz; Bruce R. McCord

2006-01-01

79

A DNA methylation fingerprint of 1628 human samples  

PubMed Central

Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases.

Fernandez, Agustin F.; Assenov, Yassen; Martin-Subero, Jose Ignacio; Balint, Balazs; Siebert, Reiner; Taniguchi, Hiroaki; Yamamoto, Hiroyuki; Hidalgo, Manuel; Tan, Aik-Choon; Galm, Oliver; Ferrer, Isidre; Sanchez-Cespedes, Montse; Villanueva, Alberto; Carmona, Javier; Sanchez-Mut, Jose V.; Berdasco, Maria; Moreno, Victor; Capella, Gabriel; Monk, David; Ballestar, Esteban; Ropero, Santiago; Martinez, Ramon; Sanchez-Carbayo, Marta; Prosper, Felipe; Agirre, Xabier; Fraga, Mario F.; Grana, Osvaldo; Perez-Jurado, Luis; Mora, Jaume; Puig, Susana; Prat, Jaime; Badimon, Lina; Puca, Annibale A.; Meltzer, Stephen J.; Lengauer, Thomas; Bridgewater, John; Bock, Christoph; Esteller, Manel

2012-01-01

80

Safeguarding forensic DNA reference samples with nullomer barcodes.  

PubMed

Unintended transfer of biological material containing DNA is a concern to all laboratories conducting PCR analysis. While forensic laboratories have protocols in place to reduce the possibility of contaminating casework samples, there is no way to detect when a reference sample is mislabeled as evidence, or contaminates a forensic sample. Thus there is public concern regarding the safeguarding of DNA submitted to crime labs. We demonstrate a method of introducing an internal amplification control to reference samples, in the form of a nullomer barcode which is based upon sequences absent or rare from publically accessible DNA databases. The detection of this barcode would indicate that the source of analyzed DNA was from a reference sample provided by an individual, and not from an evidence sample. We demonstrate that the nullomers can be added directly to collection devices (FTA paper) to allow tagging during the process of sample collection. We show that such nullomer oligonucleotides can be added to existing forensic typing and quantification kits, without affecting genotyping or quantification results. Finally, we show that even when diluted a million-fold and spilled on a knife, the nullomer tags can be clearly detected. These tags support the National Research Council of the National Academy recommendation that "Quality control procedures should be designed to identify mistakes, fraud, and bias" in forensic science (National Academy of Sciences, 2009). PMID:23756524

Goswami, Jayita; Davis, Michael C; Andersen, Tim; Alileche, Abdelkrim; Hampikian, Greg

2013-07-01

81

Proteasome-dependent degradation of replisome components regulates faithful DNA replication.  

PubMed

The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF(Pof3) (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity. PMID:23907116

Roseaulin, Laura C; Noguchi, Chiaki; Noguchi, Eishi

2013-08-15

82

Salt-tolerant phenol-degrading microorganisms isolated from Amazonian soil samples.  

PubMed

Two phenol-degrading microorganisms were isolated from Amazonian rain forest soil samples after enrichment in the presence of phenol and a high salt concentration. The yeast Candida tropicalis and the bacterium Alcaligenes faecoalis were identified using several techniques, including staining, morphological observation and biochemical tests, fatty acid profiles and 16S/18S rRNA sequencing. Both isolates, A. faecalis and C. tropicalis, were used in phenol degradation assays, with Rhodococcus erythropolis as a reference phenol-degrading bacterium, and compared to microbial populations from wastewater samples collected from phenol-contaminated environments. C. tropicalis tolerated higher concentrations of phenol and salt (16 mM and 15%, respectively) than A. faecalis (12 mM and 5.6%). The yeast also tolerated a wider pH range (3-9) during phenol degradation than A. faecalis (pH 7-9). Phenol degradation was repressed in C. tropicalis by acetate and glucose, but not by lactate. Glucose and acetate had little effect, while lactate stimulated phenol degradation in A. faecalis. To our knowledge, these soils had never been contaminated with man-made phenolic compounds and this is the first report of phenol-degrading microorganisms from Amazonian forest soil samples. The results support the idea that natural uncontaminated environments contain sufficient genetic diversity to make them valid choices for the isolation of microorganisms useful in bioremediation. PMID:11131025

Bastos, A E; Moon, D H; Rossi, A; Trevors, J T; Tsai, S M

2000-11-01

83

Development and validation of a multiplex reaction analyzing eight miniSTRs of the X chromosome for identity and kinship testing with degraded DNA.  

PubMed

We report the development of an effective system for analyzing X chromosome-linked mini short tandem repeat loci with reduced-size amplicons (less than 220 bp), useful for analyzing highly degraded DNA samples. To generate smaller amplicons, we redesigned primers for eight X-linked microsatellites (DXS7132, DXS10079, DXS10074, DXS10075, DXS6801, DXS6809, DXS6789, and DXS6799) and established efficient conditions for a multiplex PCR system (miniX). The validation tests confirmed that it has good sensitivity, requiring as little as 20 pg of DNA, and performs well with DNA from paraffin-embedded tissues, thus showing potential for improved analysis and identification of highly degraded and/or very limited DNA samples. Consequently, this system may help to solve complex forensic cases, particularly when autosomal markers convey insufficient information. PMID:23188413

Castañeda, María; Odriozola, Adrián; Gómez, Javier; Zarrabeitia, María T

2013-07-01

84

75 FR 32191 - National Health and Nutrition Examination Survey (NHANES) DNA Samples: Guidelines for Proposals...  

Federal Register 2010, 2011, 2012, 2013

...and Nutrition Examination Survey (NHANES) DNA Samples: Guidelines for Proposals To Use Samples...of describing the health of the population, DNA specimens were collected during three NHANES surveys. DNA is available in the form of crude lysates...

2010-06-07

85

28 CFR 28.13 - Analysis and indexing of DNA samples.  

Code of Federal Regulations, 2010 CFR

... 2010-07-01 false Analysis and indexing of DNA samples. 28.13 Section 28.13 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing §...

2010-07-01

86

28 CFR 28.13 - Analysis and indexing of DNA samples.  

Code of Federal Regulations, 2013 CFR

... 2013-07-01 false Analysis and indexing of DNA samples. 28.13 Section 28.13 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing §...

2013-07-01

87

Detection of Streptococcus mutans Genomic DNA in Human DNA Samples Extracted from Saliva and Blood  

PubMed Central

Caries is a multifactorial disease, and studies aiming to unravel the factors modulating its etiology must consider all known predisposing factors. One major factor is bacterial colonization, and Streptococcus mutans is the main microorganism associated with the initiation of the disease. In our studies, we have access to DNA samples extracted from human saliva and blood. In this report, we tested a real-time PCR assay developed to detect copies of genomic DNA from Streptococcus mutans in 1,424 DNA samples from humans. Our results suggest that we can determine the presence of genomic DNA copies of Streptococcus mutans in both DNA samples from caries-free and caries-affected individuals. However, we were not able to detect the presence of genomic DNA copies of Streptococcus mutans in any DNA samples extracted from peripheral blood, which suggests the assay may not be sensitive enough for this goal. Values of the threshold cycle of the real-time PCR reaction correlate with higher levels of caries experience in children, but this correlation could not be detected for adults.

Vieira, Alexandre R.; Deeley, Kathleen B.; Callahan, Nicholas F.; Noel, Jacqueline B.; Anjomshoaa, Ida; Carricato, Wendy M.; Schulhof, Louise P.; DeSensi, Rebecca S.; Gandhi, Pooja; Resick, Judith M.; Brandon, Carla A.; Rozhon, Christopher; Patir, Asli; Yildirim, Mine; Poletta, Fernando A.; Mereb, Juan C.; Letra, Ariadne; Menezes, Renato; Wendell, Steven; Lopez-Camelo, Jorge S.; Castilla, Eduardo E.; Orioli, Ieda M.; Seymen, Figen; Weyant, Robert J.; Crout, Richard; McNeil, Daniel W.; Modesto, Adriana; Marazita, Mary L.

2011-01-01

88

UV-triggered p21 degradation facilitates damaged-DNA replication and preserves genomic stability  

PubMed Central

Although many genotoxic treatments upregulate the cyclin kinase inhibitor p21, agents such as UV irradiation trigger p21 degradation. This suggests that p21 blocks a process relevant for the cellular response to UV. Here, we show that forced p21 stabilization after UV strongly impairs damaged-DNA replication, which is associated with permanent deficiencies in the recruitment of DNA polymerases from the Y family involved in translesion DNA synthesis), with the accumulation of DNA damage markers and increased genomic instability. Remarkably, such noxious effects disappear when disrupting the proliferating cell nuclear antigen (PCNA) interacting motif of stable p21, thus suggesting that the release of PCNA from p21 interaction is sufficient to allow the recruitment to PCNA of partners (such as Y polymerases) relevant for the UV response. Expression of degradable p21 only transiently delays early replication events and Y polymerase recruitment after UV irradiation. These temporary defects disappear in a manner that correlates with p21 degradation with no detectable consequences on later replication events or genomic stability. Together, our findings suggest that the biological role of UV-triggered p21 degradation is to prevent replication defects by facilitating the tolerance of UV-induced DNA lesions.

Mansilla, Sabrina F.; Soria, Gaston; Vallerga, Maria Belen; Habif, Martin; Martinez-Lopez, Wilner; Prives, Carol; Gottifredi, Vanesa

2013-01-01

89

Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology  

PubMed Central

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

90

Virology. Comment on "Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA".  

PubMed

Lucifora et al. (Research Articles, 14 March 2014, p. 1221) report that the hepatitis B virus (HBV) transcriptional template, a long-lived covalently closed circular DNA (cccDNA) molecule, is degraded noncytolytically by agents that up-regulate APOBEC3A and 3B. If these results can be independently confirmed, they would represent a critical first step toward development of a cure for the 400 million patients who are chronically infected by HBV. PMID:24926010

Chisari, Francis V; Mason, William S; Seeger, Christoph

2014-06-13

91

Microbial degradation of 2,4,6-trichloroaniline in aquatic samples and laboratory enrichment cultures  

SciTech Connect

Microorganisms present in water samples obtained from a small tributary to the Gunpowder River in Maryland degraded 2,4,6-trichloroaniline following a prolonged acclimation period. Creek water sediments, but not the co-substate aniline, reduced the lag time prior to degradation. The microorganisms in the samples could be enriched to grow 2,4,6-trichloroaniline as indicated by increase in carbon dioxide, chloride, and adenosine triphosphate and by slight biomass increases accompanying the degradation of the compound. Uptake of 2,4,6-trichloroaniline by the enrichment population was as rapid as that of the original sample population but was without an apparent lag. Similar enrichment cultures could not be developed from five other sites.

Mitchell, W.R.; Hoke, S.H.; Rosencrance, A.B.

1984-01-01

92

Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.  

PubMed

Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here, we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes ("spermatoproteasomes") contain a spermatid/sperm-specific ? subunit ?4 s/PSMA8 and/or the catalytic ? subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis. PMID:23706739

Qian, Min-Xian; Pang, Ye; Liu, Cui Hua; Haratake, Kousuke; Du, Bo-Yu; Ji, Dan-Yang; Wang, Guang-Fei; Zhu, Qian-Qian; Song, Wei; Yu, Yadong; Zhang, Xiao-Xu; Huang, Hai-Tao; Miao, Shiying; Chen, Lian-Bin; Zhang, Zi-Hui; Liang, Ya-Nan; Liu, Shan; Cha, Hwangho; Yang, Dong; Zhai, Yonggong; Komatsu, Takuo; Tsuruta, Fuminori; Li, Haitao; Cao, Cheng; Li, Wei; Li, Guo-Hong; Cheng, Yifan; Chiba, Tomoki; Wang, Linfang; Goldberg, Alfred L; Shen, Yan; Qiu, Xiao-Bo

2013-05-23

93

A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD  

Microsoft Academic Search

The homeostasis of animals is regulated not only by the growth and differentiation of cells, but also by cell death through a process known as apoptosis. Apoptosis is mediated by members of the caspase family of proteases, and eventually causes the degradation of chromosomal DNA. A caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD) have now been identified in the cytoplasmic

Masato Enari; Hideki Sakahira; Hideki Yokoyama; Katsuya Okawa; Akihiro Iwamatsu; Shigekazu Nagata

1998-01-01

94

Genomic DNA microextraction: a method to screen numerous samples.  

PubMed

Many experimental designs require the analysis of genomic DNA from a large number of samples. Although the polymerase chain reaction (PCR) can be used, the Southern blot is preferred for many assays because of its inherent reliability. The rapid acceptance of PCR, despite a significant rate of false positive/negative results, is partly due to the disadvantages of the sample preparation process for Southern blot analysis. We have devised a rapid protocol to extract high-molecular-weight genomic DNA from a large number of samples. It involves the use of a single 96-well tissue culture dish to carry out all the steps of the sample preparation. This, coupled with the use of a multichannel pipette, facilitates the simultaneous analysis of multiple samples. The procedure may be automated since no centrifugation, mixing, or transferring of the samples is necessary. The method has been used to screen embryonic stem cell clones for the presence of targeted mutations at the Hox-2.6 locus and to obtain data from human blood. PMID:1632522

Ramírez-Solis, R; Rivera-Pérez, J; Wallace, J D; Wims, M; Zheng, H; Bradley, A

1992-03-01

95

USE OF DNA:DNA COLONY HYBRIDIZATION IN THE RAPID ISOLATION OF 4-CHLOROBIPHENYL DEGRADATIVE BACTERIAL PHENOTYPES  

EPA Science Inventory

DNA:DNA colony hybridization techniques were used to select isolates from freshwater sediment samples that contain genes homologous to plasmid pSS50, coding for 4-chlorobiphenyl biodegradation. A high degree of resolution was achieved in which target organisms representing 0.3% o...

96

Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis  

Microsoft Academic Search

The role of Rad51 in an unperturbed cell cycle has been difficult to distinguish from its DNA repair function. Here, using EM to visualize replication intermediates assembled in Xenopus laevis egg extract, we show that Rad51 is required to prevent the accumulation of single-stranded DNA (ssDNA) gaps at replication forks and behind them. ssDNA gaps at forks arise from extended

Yoshitami Hashimoto; Arnab Ray Chaudhuri; Massimo Lopes; Vincenzo Costanzo

2010-01-01

97

DNA Barcoding: Error Rates Based on Comprehensive Sampling  

PubMed Central

DNA barcoding has attracted attention with promises to aid in species identification and discovery; however, few well-sampled datasets are available to test its performance. We provide the first examination of barcoding performance in a comprehensively sampled, diverse group (cypraeid marine gastropods, or cowries). We utilize previous methods for testing performance and employ a novel phylogenetic approach to calculate intraspecific variation and interspecific divergence. Error rates are estimated for (1) identifying samples against a well-characterized phylogeny, and (2) assisting in species discovery for partially known groups. We find that the lowest overall error for species identification is 4%. In contrast, barcoding performs poorly in incompletely sampled groups. Here, species delineation relies on the use of thresholds, set to differentiate between intraspecific variation and interspecific divergence. Whereas proponents envision a “barcoding gap” between the two, we find substantial overlap, leading to minimal error rates of ~17% in cowries. Moreover, error rates double if only traditionally recognized species are analyzed. Thus, DNA barcoding holds promise for identification in taxonomically well-understood and thoroughly sampled clades. However, the use of thresholds does not bode well for delineating closely related species in taxonomically understudied groups. The promise of barcoding will be realized only if based on solid taxonomic foundations.

2005-01-01

98

Sensitive digital quantification of DNA methylation in clinical samples  

PubMed Central

Abnormally methylated genes are increasingly being used as cancer biomarkers 1, 2. For clinical applications, it is important to precisely determine the number of methylated molecules in the analyzed sample. We here describe a digital approach that can enumerate one methylated molecule out of ~5000 unmethylated molecules. Individual DNA fragments can be amplified and analyzed either by flow cytometry or next generation sequencing instruments. Using methylated vimentin as a biomarker, we tested 191 plasma samples and detected cancer cases with 59% sensitivity (95% CI, 48%–70%) and 93% specificity (95% CI, 86%–97%). Using the same assay, we analyzed 80 stool samples and demonstrated 45% sensitivity for detecting colorectal adenomas (23%–68%), 41% sensitivity for detecting cancer (21%–64%), and 95% specificity (82%–99%). This digital quantification of rare methylation events should be applicable to diagnostic evaluations of clinical samples, to preclinical assessments of new epigenetic biomarkers, and to quantitative analyses of epigenetic biology.

Li, Meng; Chen, Wei-dong; Papadopoulos, Nickolas; Goodman, Steven; Bjerregaard, Niels Christian; Laurberg, S?ren; Levin, Bernard; Juhl, Hartmut; Arber, Nadir; Moinova, Helen; Durkee, Kris; Schmidt, Kerstin; He, Yiping; Diehl, Frank; Velculescu, Victor E; Zhou, Shibin; Diaz, Luis A; Kinzler, Kenneth W; Markowitz, Sanford D; Vogelstein, Bert

2010-01-01

99

Saliva samples are a viable alternative to blood samples as a source of DNA for high throughput genotyping  

PubMed Central

Background The increasing trend for incorporation of biological sample collection within clinical trials requires sample collection procedures which are convenient and acceptable for both patients and clinicians. This study investigated the feasibility of using saliva-extracted DNA in comparison to blood-derived DNA, across two genotyping platforms: Applied Biosystems TaqmanTM and Illumina BeadchipTM genome-wide arrays. Method Patients were recruited from the Pharmacogenetics of Breast Cancer Chemotherapy (PGSNPS) study. Paired blood and saliva samples were collected from 79 study participants. The Oragene DNA Self-Collection kit (DNAgenotek®) was used to collect and extract DNA from saliva. DNA from EDTA blood samples (median volume 8 ml) was extracted by Gen-Probe, Livingstone, UK. DNA yields, standard measures of DNA quality, genotype call rates and genotype concordance between paired, duplicated samples were assessed. Results Total DNA yields were lower from saliva (mean 24 ?g, range 0.2–52 ?g) than from blood (mean 210 ?g, range 58–577 ?g) and a 2-fold difference remained after adjusting for the volume of biological material collected. Protein contamination and DNA fragmentation measures were greater in saliva DNA. 78/79 saliva samples yielded sufficient DNA for use on Illumina Beadchip arrays and using Taqman assays. Four samples were randomly selected for genotyping in duplicate on the Illumina Beadchip arrays. All samples were genotyped using Taqman assays. DNA quality, as assessed by genotype call rates and genotype concordance between matched pairs of DNA was high (>97%) for each measure in both blood and saliva-derived DNA. Conclusion We conclude that DNA from saliva and blood samples is comparable when genotyping using either Taqman assays or genome-wide chip arrays. Saliva sampling has the potential to increase participant recruitment within clinical trials, as well as reducing the resources and organisation required for multicentre sample collection.

2012-01-01

100

Distortion of genetically modified organism quantification in processed foods: influence of particle size compositions and heat-induced DNA degradation.  

PubMed

Milling fractions from conventional and transgenic corn were prepared at laboratory scale and used to study the influence of sample composition and heat-induced DNA degradation on the relative quantification of genetically modified organisms (GMO) in food products. Particle size distributions of the obtained fractions (coarse grits, regular grits, meal, and flour) were characterized using a laser diffraction system. The application of two DNA isolation protocols revealed a strong correlation between the degree of comminution of the milling fractions and the DNA yield in the extracts. Mixtures of milling fractions from conventional and transgenic material (1%) were prepared and analyzed via real-time polymerase chain reaction. Accurate quantification of the adjusted GMO content was only possible in mixtures containing conventional and transgenic material in the form of analogous milling fractions, whereas mixtures of fractions exhibiting different particle size distributions delivered significantly over- and underestimated GMO contents depending on their compositions. The process of heat-induced nucleic acid degradation was followed by applying two established quantitative assays showing differences between the lengths of the recombinant and reference target sequences (A, deltal(A) = -25 bp; B, deltal(B) = +16 bp; values related to the amplicon length of the reference gene). Data obtained by the application of method A resulted in underestimated recoveries of GMO contents in the samples of heat-treated products, reflecting the favored degradation of the longer target sequence used for the detection of the transgene. In contrast, data yielded by the application of method B resulted in increasingly overestimated recoveries of GMO contents. The results show how commonly used food technological processes may lead to distortions in the results of quantitative GMO analyses. PMID:16366682

Moreano, Francisco; Busch, Ulrich; Engel, Karl-Heinz

2005-12-28

101

ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining.  

PubMed

The human disorder ataxia telangiectasia (AT), which is characterized by genetic instability and neurodegeneration, results from mutation of the ataxia telangiectasia mutated (ATM) kinase. The loss of ATM leads to cell cycle checkpoint deficiencies and other DNA damage signaling defects that do not fully explain all pathologies associated with A-T including neuronal loss. In addressing this enigma, we find here that ATM suppresses DNA double-strand break (DSB) repair by microhomology-mediated end joining (MMEJ). We show that ATM repression of DNA end-degradation is dependent on its kinase activities and that Mre11 is the major nuclease behind increased DNA end-degradation and MMEJ repair in A-T. Assessment of MMEJ by an in vivo reporter assay system reveals decreased levels of MMEJ repair in Mre11-knockdown cells and in cells treated with Mre11-nuclease inhibitor mirin. Structure-based modeling of Mre11 dimer engaging DNA ends suggests the 5' ends of a bridged DSB are juxtaposed such that DNA unwinding and 3'-5' exonuclease activities may collaborate to facilitate simultaneous pairing of extended 5' termini and exonucleolytic degradation of the 3' ends in MMEJ. Together our results provide an integrated understanding of ATM and Mre11 in MMEJ: ATM has a critical regulatory function in controlling DNA end-stability and error-prone DSB repair and Mre11 nuclease plays a major role in initiating MMEJ in mammalian cells. These functions of ATM and Mre11 could be particularly important in neuronal cells, which are post-mitotic and therefore depend on mechanisms other than homologous recombination between sister chromatids to repair DSBs. PMID:20647759

Rahal, Elias A; Henricksen, Leigh A; Li, Yuling; Williams, R Scott; Tainer, John A; Dixon, Kathleen

2010-07-15

102

ATM regulates Mre11-dependent DNA end-degradation and microhomology-mediated end joining  

PubMed Central

The human disorder ataxia telangiectasia (AT), which is characterized by genetic instability and neurodegeneration, results from mutation of the ataxia telangiectasia mutated (ATM) kinase. The loss of ATM leads to cell cycle checkpoint deficiencies and other DNA damage signaling defects that do not fully explain all pathologies associated with A-T including neuronal loss. In addressing this enigma, we find here that ATM suppresses DNA double-strand break (DSB) repair by microhomology-mediated end joining (MMEJ). We show that ATM repression of DNA end-degradation is dependent on its kinase activities and that Mre11 is the major nuclease behind increased DNA end-degradation and MMEJ repair in A-T. Assessment of MMEJ by an in vivo reporter assay system reveals decreased levels of MMEJ repair in Mre11-knockdown cells and in cells treated with Mre11-nuclease inhibitor mirin. Structure-based modeling of Mre11 dimer engaging DNA ends suggests the 5? ends of a bridged DSB are juxtaposed such that DNA unwinding and 3?–5? exonuclease activities may collaborate to facilitate simultaneous pairing of extended 5? termini and exonucleolytic degradation of the 3? ends in MMEJ. Together our results provide an integrated understanding of ATM and Mre11 in MMEJ: ATM has a critical regulatory function in controlling DNA end-stability and error-prone DSB repair and Mre11 nuclease plays a major role in initiating MMEJ in mammalian cells. These functions of ATM and Mre11 could be particularly important in neuronal cells, which are post-mitotic and therefore depend on mechanisms other than homologous recombination between sister chromatids to repair DSBs.

Rahal, Elias A; Henricksen, Leigh A; Li, Yuling; Williams, R Scott

2010-01-01

103

77 FR 34387 - National Health and Nutrition Examination Survey (NHANES) DNA Samples  

Federal Register 2010, 2011, 2012, 2013

...Nutrition Examination Survey (NHANES) DNA Samples AGENCY: Centers for Disease Control...Survey (NHANES) will not be receiving DNA proposals in the near future. NHANES is changing its plan for making DNA available for genetic research and its...

2012-06-11

104

76 FR 72417 - National Health and Nutrition Examination Survey (NHANES) DNA Samples  

Federal Register 2010, 2011, 2012, 2013

...and Nutrition Examination Survey (NHANES) DNA Samples AGENCY: Centers for Disease Control...Examination Survey (NHANES) will not be receiving DNA proposals in 2012. NHANES is changing its plan for making DNA available for genetic research and its...

2011-11-23

105

Proteotoxic stress induces a cell-cycle arrest by stimulating Lon to degrade the replication initiator DnaA.  

PubMed

The decision to initiate DNA replication is a critical step in the cell cycle of all organisms. Cells often delay replication in the face of stressful conditions, but the underlying mechanisms remain incompletely defined. Here, we demonstrate in Caulobacter crescentus that proteotoxic stress induces a cell-cycle arrest by triggering the degradation of DnaA, the conserved replication initiator. A depletion of available Hsp70 chaperone, DnaK, either through genetic manipulation or heat shock, induces synthesis of the Lon protease, which can directly degrade DnaA. Unexpectedly, we find that unfolded proteins, which accumulate following a loss of DnaK, also allosterically activate Lon to degrade DnaA, thereby ensuring a cell-cycle arrest. Our work reveals a mechanism for regulating DNA replication under adverse growth conditions. Additionally, our data indicate that unfolded proteins can actively and directly alter substrate recognition by cellular proteases. PMID:23911325

Jonas, Kristina; Liu, Jing; Chien, Peter; Laub, Michael T

2013-08-01

106

DNase 2 Is the Main DNA-Degrading Enzyme of the Stratum Corneum  

PubMed Central

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA.

Fischer, Heinz; Scherz, Jennifer; Szabo, Sandra; Mildner, Michael; Benarafa, Charaf; Torriglia, Alicia; Tschachler, Erwin; Eckhart, Leopold

2011-01-01

107

Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP.  

PubMed

The massive influx of crude oil into the Gulf of Mexico during the Deepwater Horizon (DWH) disaster triggered dramatic microbial community shifts in surface oil slick and deep plume waters. Previous work had shown several taxa, notably DWH Oceanospirillales, Cycloclasticus and Colwellia, were found to be enriched in these waters based on their dominance in conventional clone and pyrosequencing libraries and were thought to have had a significant role in the degradation of the oil. However, this type of community analysis data failed to provide direct evidence on the functional properties, such as hydrocarbon degradation of organisms. Using DNA-based stable-isotope probing with uniformly (13)C-labelled hydrocarbons, we identified several aliphatic (Alcanivorax, Marinobacter)- and polycyclic aromatic hydrocarbon (Alteromonas, Cycloclasticus, Colwellia)-degrading bacteria. We also isolated several strains (Alcanivorax, Alteromonas, Cycloclasticus, Halomonas, Marinobacter and Pseudoalteromonas) with demonstrable hydrocarbon-degrading qualities from surface slick and plume water samples collected during the active phase of the spill. Some of these organisms accounted for the majority of sequence reads representing their respective taxa in a pyrosequencing data set constructed from the same and additional water column samples. Hitherto, Alcanivorax was not identified in any of the previous water column studies analysing the microbial response to the spill and we discuss its failure to respond to the oil. Collectively, our data provide unequivocal evidence on the hydrocarbon-degrading qualities for some of the dominant taxa enriched in surface and plume waters during the DWH oil spill, and a more complete understanding of their role in the fate of the oil. PMID:23788333

Gutierrez, Tony; Singleton, David R; Berry, David; Yang, Tingting; Aitken, Michael D; Teske, Andreas

2013-11-01

108

Magnetic Scanometric DNA Microarray Detection of Methyl Tertiary Butyl Ether Degrading Bacteria for Environmental Monitoring  

PubMed Central

A magnetoresistive biosensing platform based on a single magnetic tunnel junction (MTJ) scanning probe and DNA microarrays labeled with magnetic particles has been developed to provide an inexpensive, sensitive and reliable detection of DNA. The biosensing platform was demonstrated on a DNA microarray assay for quantifying bacteria capable of degrading methyl tertiary-butyl ether (MTBE), where concentrations as low as 10 pM were detectable. Synthetic probe bacterial DNA was immobilized on a microarray glass slide surface, hybridized with the 48 base pair long biotinylated target DNA and subsequently incubated with streptavidin-coated 2.8 ?m diameter magnetic particles. The biosensing platform then makes use of a micron-sized MTJ sensor that was raster scanned across a 3 mm by 5 mm glass slide area to capture the stray magnetic field from the tagged DNA and extract two dimensional magnetic field images of the microarray. The magnetic field output is then averaged over each 100 ?m diameter DNA array spot to extract the magnetic spot intensity, analogous to the fluorescence spot intensity used in conventional optical scanners. The magnetic scanning result is compared with results from a commercial laser scanner and particle coverage optical counting to demonstrate the dynamic range and linear sensitivity of the biosensing platform as a potentially inexpensive, sensitive and portable alternative for DNA microarray detection for field applications.

Chan, Mei-Lin; Jaramillo, Gerardo; Hristova, Krassimira R.; Horsley, David A.

2010-01-01

109

The effect of geographical scale of sampling on DNA barcoding.  

PubMed

Eight years after DNA barcoding was formally proposed on a large scale, CO1 sequences are rapidly accumulating from around the world. While studies to date have mostly targeted local or regional species assemblages, the recent launch of the global iBOL project (International Barcode of Life), highlights the need to understand the effects of geographical scale on Barcoding's goals. Sampling has been central in the debate on DNA Barcoding, but the effect of the geographical scale of sampling has not yet been thoroughly and explicitly tested with empirical data. Here, we present a CO1 data set of aquatic predaceous diving beetles of the tribe Agabini, sampled throughout Europe, and use it to investigate how the geographic scale of sampling affects 1) the estimated intraspecific variation of species, 2) the genetic distance to the most closely related heterospecific, 3) the ratio of intraspecific and interspecific variation, 4) the frequency of taxonomically recognized species found to be monophyletic, and 5) query identification performance based on 6 different species assignment methods. Intraspecific variation was significantly correlated with the geographical scale of sampling (R-square = 0.7), and more than half of the species with 10 or more sampled individuals (N = 29) showed higher intraspecific variation than 1% sequence divergence. In contrast, the distance to the closest heterospecific showed a significant decrease with increasing geographical scale of sampling. The average genetic distance dropped from > 7% for samples within 1 km, to < 3.5% for samples up to > 6000 km apart. Over a third of the species were not monophyletic, and the proportion increased through locally, nationally, regionally, and continentally restricted subsets of the data. The success of identifying queries decreased with increasing spatial scale of sampling; liberal methods declined from 100% to around 90%, whereas strict methods dropped to below 50% at continental scales. The proportion of query identifications considered uncertain (more than one species < 1% distance from query) escalated from zero at local, to 50% at continental scale. Finally, by resampling the most widely sampled species we show that even if samples are collected to maximize the geographical coverage, up to 70 individuals are required to sample 95% of intraspecific variation. The results show that the geographical scale of sampling has a critical impact on the global application of DNA barcoding. Scale-effects result from the relative importance of different processes determining the composition of regional species assemblages (dispersal and ecological assembly) and global clades (demography, speciation, and extinction). The incorporation of geographical information, where available, will be required to obtain identification rates at global scales equivalent to those in regional barcoding studies. Our result hence provides an impetus for both smarter barcoding tools and sprouting national barcoding initiatives-smaller geographical scales deliver higher accuracy. PMID:22398121

Bergsten, Johannes; Bilton, David T; Fujisawa, Tomochika; Elliott, Miranda; Monaghan, Michael T; Balke, Michael; Hendrich, Lars; Geijer, Joja; Herrmann, Jan; Foster, Garth N; Ribera, Ignacio; Nilsson, Anders N; Barraclough, Timothy G; Vogler, Alfried P

2012-10-01

110

Comparison of DNA preservation methods for environmental bacterial community samples  

USGS Publications Warehouse

Field collections of environmental samples, for example corals, for molecular microbial analyses present distinct challenges. The lack of laboratory facilities in remote locations is common, and preservation of microbial community DNA for later study is critical. A particular challenge is keeping samples frozen in transit. Five nucleic acid preservation methods that do not require cold storage were compared for effectiveness over time and ease of use. Mixed microbial communities of known composition were created and preserved by DNAgard™, RNAlater®, DMSO–EDTA–salt (DESS), FTA® cards, and FTA Elute® cards. Automated ribosomal intergenic spacer analysis and clone libraries were used to detect specific changes in the faux communities over weeks and months of storage. A previously known bias in FTA® cards that results in lower recovery of pure cultures of Gram-positive bacteria was also detected in mixed community samples. There appears to be a uniform bias across all five preservation methods against microorganisms with high G + C DNA. Overall, the liquid-based preservatives (DNAgard™, RNAlater®, and DESS) outperformed the card-based methods. No single liquid method clearly outperformed the others, leaving method choice to be based on experimental design, field facilities, shipping constraints, and allowable cost.

Gray, Michael A.; Pratte, Zoe A.; Kellogg, Christina A.

2013-01-01

111

Detection of Helicobacter pylori DNA in Fecal Samples from Infected Individuals  

Microsoft Academic Search

Stool, gastric biopsy, and serum samples were collected from 22 subjects. DNA from stool was extracted, amplified, and hybridized with primers specific for the 16S rRNA gene of Helicobacter pylori. DNA from gastric biopsy specimens was analyzed similarly for comparison. Universal primers were used to confirm successful extraction of DNA from samples. Histologic, serologic, and DNA analyses were scored in

WILLIAM A. GRAMLEY; ALI ASGHAR; HENRY F. FRIERSON; STEVEN M. POWELL

1999-01-01

112

Identification of Hydrocarbon-Degrading Bacteria in Soil by Reverse Sample Genome Probing  

Microsoft Academic Search

Bacteria with limited genomic cross-hybridization were isolated from soil contaminated with C51, a mixture of hydrocarbons, and identified by partial 16S rRNA sequencing. Filters containing denatured genomic DNAs were used in a reverse sample genome probe (RSGP) procedure for analysis of the effect of an easily degradable compound (toluene) and a highly recalcitrant compound (dicyclopentadiene (DCPD)) on community compo- sition.

YIN SHEN; LESTER G. STEHMEIER; GERRIT VOORDOUW

1998-01-01

113

PCNA-coupled p21 degradation after DNA damage: the exception that confirms the rule?  

PubMed Central

While many are the examples of DNA damaging treatments that induce p21 accumulation, the conception of p21 upregulation as the universal response to genotoxic stress has come to an end. Compelling evidences have demonstrated the existence of converging signals that negatively regulate p21 bellow basal levels when replication forks are blocked. Moreover, conclusive reports identified the E3-ligase CRL4CDT2 (CUL4-DDB1-CDT2) as the enzymatic complex that promotes p21 proteolysis when treatments such as UV irradiation trigger replication fork stress. A pre-requisite for CRL4CDT2-driven proteolysis is the interaction of p21 with PCNA. Interestingly as well, CRL4CDT2-dependent proteolysis is not limited to p21 and affects other PCNA partners, including the specialized DNA polymerase ?(pol eta). These recent discoveries are particularly intriguing since the UV-induced degradation of p21 has been shown to be required for efficient pol ? recruitment to DNA lesions. Herein we review the findings that lead to the identification of the molecular mechanism that triggers damage-induced PCNA-coupled protein proteolysis. We propose a novel model in which CRL4CDT2-dependent protein degradation facilitates a sequential and dynamic exchange between PIP box-bearing proteins at stall forks during Translesion DNA synthesis (TLS). Moreover, given the tight spatiotemporal control that CRL4CDT2-driven proteolysis is able to confer to PCNA-regulated processes, we discuss the impact that this degradation mechanism might have in other molecular switches associated with the repair of damaged DNA.

Soria, Gaston; Gottifredi, Vanesa

2010-01-01

114

Feasibility of High-Throughput Genome-Wide Genotyping using DNA from Stored Buccal Cell Samples.  

PubMed

It is unclear if buccal cell samples contain sufficient human DNA with adequately sized fragments for high throughput genetic bioassays. Yet buccal cell sample collection is an attractive alternative to gathering blood samples for genetic epidemiologists engaged in large-scale genetic biomarker studies. We assessed the genotyping efficiency (GE) and genotyping concordance (GC) of buccal cell DNA samples compared to corresponding blood DNA samples, from 32 Nurses' Health Study (NHS) participants using the Illumina Infinium 660W-Quad platform. We also assessed how GE and GC accuracy varied as a function of DNA concentration using serial dilutions of buccal DNA samples. Finally we determined the nature and genomic distribution of discordant genotypes in buccal DNA samples. The mean GE of undiluted buccal cell DNA samples was high (99.32%), as was the GC between the paired buccal and blood samples (99.29%). GC between the dilutions versus the undiluted buccal DNA was also very high (>97%), though both GE and GC notably declined at DNA concentrations less than 5 ng/mul. Most (>95%) genotype determinations in buccal cell samples were of the "missing call" variety (as opposed to the "alternative genotype call" variety) across the spectrum of buccal DNA concentrations studied. Finally, for buccal DNA concentration above 1.7 ng/ul, discordant genotyping calls did not cluster in any particular chromosome. Buccal cell-derived DNA represents a viable alternative to blood DNA for genotyping on a high-density platform. PMID:20520743

Loomis, Stephanie J; Olson, Lana M; Pasquale, Louis R; Wiggs, Janey; Mirel, Daniel; Crenshaw, Andrew; Parkin, Melissa; Rahhal, Brandon; Tetreault, Stephanie; Kraft, Peter; Tworoger, Shelley S; Haines, Jonathan L; Kang, Jae H

2010-01-01

115

Capacity of Nine Thermostable DNA Polymerases To Mediate DNA Amplification in the Presence of PCR-Inhibiting Samples  

Microsoft Academic Search

The PCR is an extremely powerful method for detecting microorganisms. However, its full potential as a rapid detection method is limited by the inhibition of the thermostable DNA polymerase from Thermus aqua- ticus by many components found in complex biological samples. In this study, we have compared the effects of known PCR-inhibiting samples on nine thermostable DNA polymerases. Samples of

WALEED ABU AL-SOUD; PETER RÅDSTROM

1998-01-01

116

Strain dependent UV degradation of Escherichia coli DNA monitored by Fourier transform infrared spectroscopy.  

PubMed

In this work we present a method for detection of DNA isolated from nonpathogenic Escherichia coli strains, respectively. Untreated and UV irradiated bacterial DNAs were analyzed by FT-IR spectroscopy, to investigate their screening characteristic features and their structural radiotolerance at 253.7nm. FT-IR spectra, providing a high molecular structural information, have been analyzed in the wavenumber range 800-1800cm(-1). FT-IR signatures, spectroscopic band assignments and structural interpretations of these DNAs are reported. Also, UV damage at the DNA molecular level is of interest. Strain dependent UV degradation of DNA from E. coli has been observed. Particularly, alterations in nucleic acid bases, base pairing and base stacking have been found. Also changes in the DNA conformation and deoxyribose were detected. Based on this work, specific E. coli DNA-ligand interactions, drug development and vaccine design for a better understanding of the infection mechanism caused by an interference between pathogenic and nonpathogenic bacteria and for a better control of disease, respectively, might be further investigated using Fourier transform infrared spectroscopy. Besides, understanding the pathways for UV damaged DNA response, like nucleic acids repair mechanisms is appreciated. PMID:24333761

Muntean, Cristina M; Lapusan, Alexandra; Mihaiu, Liora; Stefan, Razvan

2014-01-01

117

DNA-based stable isotope probing coupled with cultivation methods implicates Methylophaga in hydrocarbon degradation.  

PubMed

Marine hydrocarbon-degrading bacteria perform a fundamental role in the oxidation and ultimate removal of crude oil and its petrochemical derivatives in coastal and open ocean environments. Those with an almost exclusive ability to utilize hydrocarbons as a sole carbon and energy source have been found confined to just a few genera. Here we used stable isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate hydrocarbon-degrading bacteria in coastal North Carolina sea water (Beaufort Inlet, USA) with uniformly labeled [(13)C]n-hexadecane. The dominant sequences in clone libraries constructed from (13)C-enriched bacterial DNA (from n-hexadecane enrichments) were identified to belong to the genus Alcanivorax, with ?98% sequence identity to the closest type strain-thus representing a putative novel phylogenetic taxon within this genus. Unexpectedly, we also identified (13)C-enriched sequences in heavy DNA fractions that were affiliated to the genus Methylophaga. This is a contentious group since, though some of its members have been proposed to degrade hydrocarbons, substantive evidence has not previously confirmed this. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Alcanivorax and Methylophaga to determine their abundance in incubations amended with unlabeled n-hexadecane. Both showed substantial increases in gene copy number during the experiments. Subsequently, we isolated a strain representing the SIP-identified Methylophaga sequences (99.9% 16S rRNA gene sequence identity) and used it to show, for the first time, direct evidence of hydrocarbon degradation by a cultured Methylophaga sp. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of hydrocarbon-degrading bacteria in the marine environment. PMID:24578702

Mishamandani, Sara; Gutierrez, Tony; Aitken, Michael D

2014-01-01

118

DNA-based stable isotope probing coupled with cultivation methods implicates Methylophaga in hydrocarbon degradation  

PubMed Central

Marine hydrocarbon-degrading bacteria perform a fundamental role in the oxidation and ultimate removal of crude oil and its petrochemical derivatives in coastal and open ocean environments. Those with an almost exclusive ability to utilize hydrocarbons as a sole carbon and energy source have been found confined to just a few genera. Here we used stable isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate hydrocarbon-degrading bacteria in coastal North Carolina sea water (Beaufort Inlet, USA) with uniformly labeled [13C]n-hexadecane. The dominant sequences in clone libraries constructed from 13C-enriched bacterial DNA (from n-hexadecane enrichments) were identified to belong to the genus Alcanivorax, with ?98% sequence identity to the closest type strain—thus representing a putative novel phylogenetic taxon within this genus. Unexpectedly, we also identified 13C-enriched sequences in heavy DNA fractions that were affiliated to the genus Methylophaga. This is a contentious group since, though some of its members have been proposed to degrade hydrocarbons, substantive evidence has not previously confirmed this. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Alcanivorax and Methylophaga to determine their abundance in incubations amended with unlabeled n-hexadecane. Both showed substantial increases in gene copy number during the experiments. Subsequently, we isolated a strain representing the SIP-identified Methylophaga sequences (99.9% 16S rRNA gene sequence identity) and used it to show, for the first time, direct evidence of hydrocarbon degradation by a cultured Methylophaga sp. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of hydrocarbon-degrading bacteria in the marine environment.

Mishamandani, Sara; Gutierrez, Tony; Aitken, Michael D.

2014-01-01

119

Degradation of DNA and structure-activity relationship between bleomycins A sub 2 and B sub 2 in the absence of DNA repair  

SciTech Connect

The contribution of DNA repair to the net number of DNA breaks produced during chemical degradation of DNA was determined by using temperature-sensitive mutant cells deficient in ATP-dependent DNA ligase. In a very sensitive assay for determining lesions introduced into Saccharomyces cerevisiae DNAs, 2-{sup 14}C- and 6-{sup 3}H-prelabeled DNAs from ligase-proficient and ligase-deficient cells were sedimented together through precalibrated, isokinetic alkaline sucrose gradients. DNA ligation was slower after chemical degradation of DNA by bleomycin than after {gamma} irradiation. DNA breaks increased approximately linearly with drug concentrations, and were approximately equivalent for ligase-proficient and ligase-deficient cells. These results were unexpected because ligase-deficient, but not ligase-proficient, cells lacked the capacity to eliminate DNA breaks produced by bleomycin. The results indicated that DNA repair did not occur during the chemical degradation of DNA under the experimental conditions. Bleomycin B{sub 2} produced considerably more DNA breaks than bleomycin A{sub 2} over a range of concentrations in ligase-proficient cells, which tolerated higher numbers of DNA breaks in general than ligase-deficient cells. The chemical analogues are structurally identical except for their cationic C-terminal amine. The actual number of DNA breaks produced by bleomycin A{sub 2} or bleomycin B{sub 2}, and not the concentration of bleomycin A{sub 2} or bleomycin B{sub 2} per se, determined the amount of cell killing. DNA repair is critical in quantitating DNA breaks produced by chemicals, but was ruled out as a factor in the higher DNA breakage by bleomycin B{sub 2} than bleomycin A{sub 2}.

Moore, C.W. (City Univ. of New York, NY (USA))

1990-02-06

120

Degradation of p12 subunit by CRL4Cdt2 E3 ligase inhibits fork progression after DNA damage.  

PubMed

After acute DNA damage, the cell arrests S-phase progression by inhibiting origin initiation and fork progression to repair damaged DNA. The intra-S-phase checkpoint kinase Chk1 phosphorylates Cdc25A to target the latter for degradation by CRL1(?-TrCP) and so inhibit origin firing. The mechanism for inhibiting fork progression, however, has not been identified. Here, we show that degradation of p12, the fourth subunit of DNA polymerase ?, is critical for inhibiting fork progression. CRL4(Cdt2) is an E3 ligase that ubiquitinates and degrades p12 after UV treatment. Cells expressing a stable form of p12 exhibit UV-resistant DNA synthesis. DNA fiber assay and alkaline-sucrose gradient assay demonstrate that the impairment of fork progression after DNA damage requires p12 degradation. These results suggest that ubiquitination of p12 through CRL4(Cdt2) and subsequent degradation form one mechanism by which a cell responds to DNA damage to inhibit fork progression. PMID:24022480

Terai, Kenta; Shibata, Etsuko; Abbas, Tarek; Dutta, Anindya

2013-10-18

121

Factors leading to the degradation/loss of insulin in postmortem blood samples.  

PubMed

Since lethal insulin injection has been used in murder and suicide cases, its non-ambiguous detection in postmortem, mostly hemolytic blood samples is still a problem. In the present study the stability of insulin and reasons for its loss in those blood samples were examined. When incubated with buffer, serum or with intact blood cell suspensions insulin concentrations were found to remain stable over time, but a significant loss of insulin was observed in hemolyzed blood samples. This was not due to an enzymatic cleavage, but predominantly to the presence of hemoglobin. Incubation of insulin with a hemoglobin solution containing the same hemoglobin content as hemolyzed blood caused a dramatic decrease of the insulin concentration. Degradation of insulin reached its maximum after 23h of incubation. The charge state of the ferric ion of hemoglobin could not be held accountable for the insulin-loss, but rather the protein part of hemoglobin. Alkylation experiments using iodoacetamide suggested that the thiol groups of the globin molecule are involved in the insulin loss preventing degradation at least partially. The same was observed by lowering the pH to 2.7 in the incubation mixture. Two degradation products of insulin were identified by mass spectrometry such as modified insulin A and B chains with 4 (A chain) and 2Da (B chain) lower masses. These results suggest that thiol groups of hemoglobin cause splitting of the disulfide bonds of insulin which immediately leads to the formation of new intramolecular disulfide bridges, a reaction which occurs in hemolytic blood and may explain the gradual loss of insulin in postmortem blood samples. PMID:24973725

Wunder, Cora; Kauert, Gerold F; Toennes, Stefan W

2014-08-01

122

Critical points of DNA quantification by real-time PCR - effects of DNA extraction method and sample matrix on quantification of genetically modified organisms  

PubMed Central

Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated. Conclusion The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR.

Cankar, Katarina; Stebih, Dejan; Dreo, Tanja; Zel, Jana; Gruden, Kristina

2006-01-01

123

DNA damage accumulation and TRF2 degradation in atypical Werner syndrome fibroblasts with LMNA mutations  

PubMed Central

Segmental progeroid syndromes are groups of disorders with multiple features suggestive of accelerated aging. One subset of adult-onset progeroid syndromes, referred to as atypical Werner syndrome, is caused by mutations in the LMNA gene, which encodes a class of nuclear intermediate filaments, lamin A/C. We previously described rapid telomere attrition and accelerated replicative senescence in cultured fibroblasts overexpressing mutant lamin A. In this study, we investigated the cellular phenotypes associated with accelerated telomere shortening in LMNA mutant primary fibroblasts. In early passage primary fibroblasts with R133L or L140R LMNA mutations, shelterin protein components were already reduced while cells still retained telomere lengths comparable to those of controls. There was a significant inverse correlation between the degree of abnormal nuclear morphology and the level of TRF2, a shelterin subunit, suggesting a potential causal relationship. Stabilization of the telomeres via the introduction of the catalytic subunit of human telomerase, hTERT (human telomerase reverse transcriptase), did not prevent degradation of shelterin components, indicating that reduced TRF2 in LMNA mutants is not mediated by short telomeres. Interestingly, ?-H2AX foci (reflecting double strand DNA damage) in early passage LMNA mutant primary fibroblasts and LMNA mutant hTERT fibroblasts were markedly increased in non-telomeric regions of DNA. Our results raise the possibility that mutant lamin A/C causes global genomic instability with accumulation of non-telomeric DNA damage as an early event, followed by TRF2 degradation and telomere shortening.

Saha, Bidisha; Zitnik, Galynn; Johnson, Simon; Nguyen, Quyen; Risques, Rosa A.; Martin, George M.; Oshima, Junko

2013-01-01

124

METAL CONTAMINANTS PROMOTE DEGRADATION OF LIPID/DNA COMPLEXES DURING LYOPHILIZATION  

PubMed Central

SUMMARY Oxidation reactions represent an important degradation pathway of nucleic acid-based pharmaceuticals. To evaluate the role of metal contamination and chelating agents in the formation of reactive oxygen species (ROS) during lyophilization, ROS generation and the stability of lipid/DNA complexes were investigated. Trehalose-containing formulations were lyophilized with different levels of transition metals. ROS generation was examined by adding proxyl fluorescamine to the formulations prior to freeze-drying. Results show that ROS were generated during lyophilization, and both supercoil content and transfection rates decreased as the levels of metal-induced ROS increased. The experiments incorporating chelators demonstrated that some of these agents (e.g., DTPA, desferal) clearly suppress ROS generation, while others (e.g., EDTA) enhance ROS. Surprisingly, there was not a strong correlation of ROS generated in the presence of chelators with the maintenance of supercoil content. In this study, we demonstrated the adverse effects of the presence of metals (especially Fe2+) in nonviral vector formulations. While some chelators attenuate ROS generation and preserve DNA integrity, the effects of these additives on vector stability during lyophilization are difficult to predict. Further study is needed to develop potent formulation strategies that inhibit ROS generation and DNA degradation during lyophilization and storage.

Molina, Marion d.C.; Anchordoquy, Thomas J.

2007-01-01

125

Norcantharidin inhibits DNA replication and induces mitotic catastrophe by degrading initiation protein Cdc6.  

PubMed

Cdc6, an essential initiation protein for DNA replication, also participates in the ATR checkpoint pathway and plays a vital role in tumorigenesis. It is involved in the androgen receptor (AR) signal transduction and promotes the malignant progression of prostate cancer (PCa). In this study, we report that norcantharidin (NCTD) induces the degradation of Cdc6 in DU145 PCa cells and as a result, the assembly of pre-replication complexes (pre-RCs) was disturbed and DNA replication was inhibited. Furthermore, treatment with NCTD blocked ATR binding to chromatin and the cells progressed into mitosis under stress induced by hydroxyurea (HU), indicating that the ATR checkpoint was evaded. Aberrant mitosis and hence, apoptosis were also observed following treatment with NCTD. Finally, NCTD exerted strong synergistic cytotoxic effects in combination with another mitotic inhibitor, paclitaxel, [combination index (CI <1)]. These data suggest that NCTD not only inhibits DNA replication but also disables the ATR-dependent checkpoint pathway by inducing Cdc6 degradation, which leads to mitotic catastrophe in DU145 cells. These findings also provide a promising prospect for the combination treatment of paclitaxel and NCTD or Cdc6 deletion in PCa. PMID:23612688

Chen, Sansan; Wan, Pei; Ding, Wen; Li, Fei; He, Chengwu; Chen, Pengliang; Li, Hongwei; Hu, Zhiming; Tan, Wanlong; Li, Jinlong

2013-07-01

126

DNA-SIP Reveals That Syntrophaceae Play an Important Role in Methanogenic Hexadecane Degradation  

PubMed Central

The methanogenic degradation of linear alkanes is a common process in oil-impacted environments. However, little is known about the key players involved in this process. Here, the hexadecane-degrading organisms in a methanogenic, hexadecane-degrading consortium designated M82 obtained from Shengli oilfield and maintained at 35°C for over 4 years, were identified by DNA-stable isotope probing with UL-13C-hexadecane, followed by density-resolved terminal restriction fragment length polymorphism (T-RFLP) analysis, cloning and phylogenetic analysis of 16S rRNA gene fragments. Compared to the fractions of the 12C treatment, the relative abundance of two phylotypes significantly increased in the heavy fractions of the 13C-hexadecane incubated microcosm. One belongs to a uncultured member of the bacterial family Syntrophaceae, which show 95–97% rRNA sequence identity with Smithella propionica, and the other is affiliated with Methanoculleus receptaculi (>99% sequence identity). The results of the present study prove the significant role of uncultured Syntrophaceae in degradation of hexadecane, probably through syntrophic interactions with hydrogenotrophic methanogens.

Cheng, Lei; Ding, Chen; Li, Qiang; He, Qiao; Dai, Li-rong; Zhang, Hui

2013-01-01

127

In situ studies on the time-dependent degradation of recombinant corn DNA and protein in the bovine rumen.  

PubMed

An in situ technique was adopted to investigate the time-dependent ruminal degradation of chloroplast compared with recombinant DNA of Bt176 corn using conventional and quantitative PCR assays. In parallel, the Cry1Ab protein content and fragment sizes were determined by ELISA and immunoblotting techniques. Triplicate nylon bags filled with 5 g of each substrate (whole-plant isogenic, whole-plant transgenic, ensiled isogenic, and ensiled transgenic corn) were positioned within the rumen of 5 rumen-cannulated, nonlactating cows and incubated for 2, 4, 8, 16, 24, and 48 h. To investigate the DNA degradation process, PCR assays were developed to detect fragments of the endogenous highly abundant rubisco gene (173, 896, 1,197, and 1,753 bp) and of the recombinant cry1Ab gene (211, 420, 727, and 1,423 bp). Short fragments of rubisco (<431 bp) and cry1Ab DNA (211 bp) were amplifiable in whole-plant and ensiled corn samples incubated in the rumen for 48 h, whereas the traceability of larger fragments depended on previous processing of the sample (whole-plant or ensiled corn), the length of the target sequence, and concomitantly on the length of time incubated in the rumen. Quantification of rubisco and cry1Ab gene fragments applying real-time PCR assays revealed degradation to <20% of initial 0-h values within 2 h and <0.5% after 48 h of ruminal incubation. Analysis of Cry1Ab protein in whole-plant corn using the ELISA technique revealed a decrease to 28.0% of the initial value within 2 h and to 2.6% within 48 h. The concentration of Cry1Ab protein of ensiled corn was only 10% that of whole-plant corn. Ensiled corn Cry1Ab protein decreased to 10% of initial values after 48 h of ruminal incubation. Using an immunoblotting technique, the full-size Cry1Ab protein was only detectable up to 8 h; thereafter, only fragments of approximately 17 and 34 kDa size were found. In conclusion, ruminal digestion decreased the presence of functional cry1Ab gene fragments. It is unlikely that full-size, functional Cry1Ab protein will be present after 8 h of incubation in the rumen. Therefore, results based on ELISA measurements should be interpreted carefully and verified by another detection method that discriminates between the full-size and fragmented Cry1Ab protein. PMID:16361500

Wiedemann, S; Lutz, B; Kurtz, H; Schwarz, F J; Albrecht, C

2006-01-01

128

Time-Resolved DNA Stable Isotope Probing Links Desulfobacterales- and Coriobacteriaceae-Related Bacteria to Anaerobic Degradation of Benzene under Methanogenic Conditions  

PubMed Central

To identify the microorganisms involved in benzene degradation, DNA-stable isotope probing (SIP) with 13C-benzene was applied to a methanogenic benzene-degrading enrichment culture. Pyrosequencing of ribosomal RNA (rRNA) gene sequences revealed that the community structure was highly complex in spite of a 3-year incubation only with benzene. The culture degraded 98% of approximately 1 mM 13C-benzene and mineralized 72% of that within 63 d. The terminal restriction fragment length polymorphism (T-RFLP) profiles of the buoyant density fractions revealed the incorporation of 13C into two phylotypes after 64 d. These two phylotypes were determined to be Desulfobacterales- and Coriobacteriaceae-related bacteria by cloning and sequencing of the 16S rRNA gene in the 13C-labeled DNA abundant fraction. Comparative pyrosequencing analysis of the buoyant density fractions of 12C- and 13C-labeled samples indicated the incorporation of 13C into three bacterial and one archaeal OTUs related to Desulfobacterales, Coriobacteriales, Rhodocyclaceae, and Methanosarcinales. The first two OTUs included the bacteria detected by T-RFLP-cloning-sequencing analysis. Furthermore, time-resolved SIP analysis confirmed that the activity of all these microbes appeared at the earliest stage of degradation. In this methanogenic culture, Desulfobacterales- and Coriobacteriaceae-related bacteria were most likely to be the major benzene degraders.

Noguchi, Mana; Kurisu, Futoshi; Kasuga, Ikuro; Furumai, Hiroaki

2014-01-01

129

An evaluation of long-term preservation methods for brown bear (Ursus arctos) faecal DNA samples  

USGS Publications Warehouse

Relatively few large-scale faecal DNA studies have been initiated due to difficulties in amplifying low quality and quantity DNA template. To improve brown bear faecal DNA PCR amplification success rates and to determine post collection sample longevity, five preservation methods were evaluated: 90% ethanol, DETs buffer, silica-dried, oven-dried stored at room temperature, and oven-dried stored at -20??C. Preservation effectiveness was evaluated for 50 faecal samples by PCR amplification of a mitochondrial DNA (mtDNA) locus (???146 bp) and a nuclear DNA (nDNA) locus (???200 bp) at time points of one week, one month, three months and six months. Preservation method and storage time significantly impacted mtDNA and nDNA amplification success rates. For mtDNA, all preservation methods had ??? 75% success at one week, but storage time had a significant impact on the effectiveness of the silica preservation method. Ethanol preserved samples had the highest success rates for both mtDNA (86.5%) and nDNA (84%). Nuclear DNA amplification success rates ranged from 26-88%, and storage time had a significant impact on all methods but ethanol. Preservation method and storage time should be important considerations for researchers planning projects utilizing faecal DNA. We recommend preservation of faecal samples in 90% ethanol when feasible, although when collecting in remote field conditions or for both DNA and hormone assays a dry collection method may be advantageous.

Murphy, M. A.; Waits, L. P.; Kendall, K. C.; Wasser, S. K.; Higbee, J. A.; Bogden, R.

2002-01-01

130

Degradable and biocompatible poly(N,N-dimethylaminoethyl methacrylate-co-caprolactone)s as DNA transfection agents.  

PubMed

This study describes the synthesis of a set of novel, degradable block copolymers for DNA transfection, and analyzes their physicochemical and biological properties. PEO macro-azoinitiators are used for the free radical copolymerization of DMAEMA and MDO, resulting in a series of different quaternized or non-quaternized block copolymers. All of the polymers show little cytotoxicity and full degradability, and thus, based on their favorable properties, may represent promising vectors for in vivo applications. Marked differences in DNA complexation efficacies and biological activities are observed, and one of the poly(PEG-co-(MDO-co-DMAEMA))s is identified as optimal for DNA transfection. PMID:23682036

Zhang, Yi; Aigner, Achim; Agarwal, Seema

2013-09-01

131

Effect of DNA extraction and sample preservation method on rumen bacterial population.  

PubMed

The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile. PMID:24125910

Fliegerova, Katerina; Tapio, Ilma; Bonin, Aurelie; Mrazek, Jakub; Callegari, Maria Luisa; Bani, Paolo; Bayat, Alireza; Vilkki, Johanna; Kope?ný, Jan; Shingfield, Kevin J; Boyer, Frederic; Coissac, Eric; Taberlet, Pierre; Wallace, R John

2014-10-01

132

Method and apparatus for transport, introduction, atomization and excitation of emission spectrum for quantitative analysis of high temperature gas sample streams containing vapor and particulates without degradation of sample stream temperature  

DOEpatents

A sample transport, sample introduction, and flame excitation system for spectrometric analysis of high temperature gas streams which eliminates degradation of the sample stream by condensation losses.

Eckels, David E. (Ankeny, IA); Hass, William J. (Ames, IA)

1989-05-30

133

Phosphorylation of human TFAM in mitochondria impairs DNA binding and promotes degradation by the AAA+ Lon protease  

PubMed Central

SUMMARY Human mitochondrial transcription factor A (TFAM) is a high-mobility group (HMG) protein at the nexus of mitochondrial DNA (mtDNA) replication, transcription and inheritance. Little is known about the mechanisms underlying its post-translational regulation. Here, we demonstrate that TFAM is phosphorylated within its HMG box 1 (HMG1) by cAMP-dependent protein kinase in mitochondria. HMG1 phosphorylation impairs the ability of TFAM to bind DNA and to activate transcription. We show that only DNA-free TFAM is degraded by the Lon protease, which is inhibited by the anti-cancer drug bortezomib. In cells with normal mtDNA levels, HMG1-phosphorylated TFAM is degraded by Lon. However in cells with severe mtDNA deficits, non-phosphorylated TFAM is also degraded as it is DNA-free. Depleting Lon in these cells increases TFAM, and upregulates mtDNA content, albeit transiently. Phosphorylation and proteolysis thus provide mechanisms for rapidly fine-tuning TFAM function and abundance in mitochondria, which are crucial for maintaining and expressing mtDNA.

Lu, Bin; Lee, Jae; Nie, Xiaobo; Li, Min; Morozov, Yaroslav I.; Venkatesh, Sundararajan; Bogenhagen, Daniel F.; Temiakov, Dmitry; Suzuki, Carolyn K.

2013-01-01

134

Phosphorylation of human TFAM in mitochondria impairs DNA binding and promotes degradation by the AAA+ Lon protease.  

PubMed

Human mitochondrial transcription factor A (TFAM) is a high-mobility group (HMG) protein at the nexus of mitochondrial DNA (mtDNA) replication, transcription, and inheritance. Little is known about the mechanisms underlying its posttranslational regulation. Here, we demonstrate that TFAM is phosphorylated within its HMG box 1 (HMG1) by cAMP-dependent protein kinase in mitochondria. HMG1 phosphorylation impairs the ability of TFAM to bind DNA and to activate transcription. We show that only DNA-free TFAM is degraded by the Lon protease, which is inhibited by the anticancer drug bortezomib. In cells with normal mtDNA levels, HMG1-phosphorylated TFAM is degraded by Lon. However, in cells with severe mtDNA deficits, nonphosphorylated TFAM is also degraded, as it is DNA free. Depleting Lon in these cells increases levels of TFAM and upregulates mtDNA content, albeit transiently. Phosphorylation and proteolysis thus provide mechanisms for rapid fine-tuning of TFAM function and abundance in mitochondria, which are crucial for maintaining and expressing mtDNA. PMID:23201127

Lu, Bin; Lee, Jae; Nie, Xiaobo; Li, Min; Morozov, Yaroslav I; Venkatesh, Sundararajan; Bogenhagen, Daniel F; Temiakov, Dmitry; Suzuki, Carolyn K

2013-01-10

135

Interpretation guidelines for multilocus STR forensic profiles from low template DNA samples.  

PubMed

Low template (LT) DNA testing is a more sensitive method of PCR DNA typing which tests lower quantities of DNA compared to traditional PCR DNA protocols. Methods applied in this testing involve amplification or postamplification efforts to increase detection sensitivity. Establishing the interpretation rules of the results obtained is condition sine qua non for successful incorporation of this valuable technique into forensic casework. Here we describe a successfully optimized and validated approach to interpretation of LT-DNA samples. PMID:22139662

Budimlija, Zoran M; Caragine, Theresa A

2012-01-01

136

Mechanisms of lifetime degradation in Si/ARC samples patterned by laser lift-off  

NASA Astrophysics Data System (ADS)

Applications for laser patterning in Si photovoltaics include (i) patterning SiO2 or SiN layers with openings for local contacts and (ii) laser-doped selective emitter (LDSE) processes, in which the contact open is accompanied by the diffusion of dopants into a locally melted Si area. While contact open processes are best performed with UV wavelengths that can be strongly absorbed by the SiN or SiO2 (allowing layer ablation with a minimum of Si heating), the Si melt depth required by LDSE requires irradiation at longer laser wavelengths where these antireflection coatings (ARCs) no longer absorb well. An optimized LDSE process must thus produce Si melting as well as the least amount of Si vaporization sufficient to lift off the overlying ARC. In this work, we investigate the mechanisms for lifetime degradation in Si(p-type, 100-oriented)/ARC samples resulting from 20 ns pulsed laser irradiation at 532 nm at fluences near the threshold for ARC removal. To differentiate between lifetime degradation induced by changes in the passivation layer vs. changes in the Si itself, samples were lifetime mapped after patterned laser irradiation and then again after a wet ARC strip and repassivation. Samples with ARCs of thermal SiO2 and PECVD SiN typically showed some residual Si damage after irradiation at fluences sufficient for contact open. Interestingly, irradiation of the SiO2 samples at lower fluences, between the threshold for Si melting and ARC removal, showed damage to the SiO2 passivation, but no residual Si damage. Explanations for these observations and related results will be discussed.

Saenger, K. L.

2012-10-01

137

DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment.  

PubMed

Global groundwater resources are constantly challenged by a multitude of contaminants such as aromatic hydrocarbons. Especially in anaerobic habitats, a large diversity of unrecognized microbial populations may be responsible for their degradation. Still, our present understanding of the respective microbiota and their ecophysiology is almost exclusively based on a small number of cultured organisms, mostly within the Proteobacteria. Here, by DNA-based stable isotope probing (SIP), we directly identified the most active sulfate-reducing toluene degraders in a diverse sedimentary microbial community originating from a tar-oil-contaminated aquifer at a former coal gasification plant. On incubation of fresh sediments with (13)C(7)-toluene, the production of both sulfide and (13)CO(2) was clearly coupled to the (13)C-labeling of DNA of microbes related to Desulfosporosinus spp. within the Peptococcaceae (Clostridia). The screening of labeled DNA fractions also suggested a novel benzylsuccinate synthase alpha-subunit (bssA) sequence type previously only detected in the environment to be tentatively affiliated with these degraders. However, carbon flow from the contaminant into degrader DNA was only ?50%, pointing toward high ratios of heterotrophic CO(2)-fixation during assimilation of acetyl-CoA originating from the contaminant by these degraders. These findings demonstrate that the importance of non-proteobacterial populations in anaerobic aromatics degradation, as well as their specific ecophysiology in the subsurface may still be largely ungrasped. PMID:20428224

Winderl, Christian; Penning, Holger; Netzer, Frederick von; Meckenstock, Rainer U; Lueders, Tillmann

2010-10-01

138

cDNA hybrid capture improves transcriptome analysis on low-input and archived samples.  

PubMed

The use of massively parallel sequencing for studying RNA expression has greatly enhanced our understanding of the transcriptome through the myriad ways these data can be characterized. In particular, clinical samples provide important insights about RNA expression in health and disease, yet these studies can be complicated by RNA degradation that results from the use of formalin as a clinical preservative and by the limited amounts of RNA often available from these precious samples. In this study we describe the combined use of RNA sequencing with an exome capture selection step to enhance the yield of on-exon sequencing read data when compared with RNA sequencing alone. In particular, the exome capture step preserves the dynamic range of expression, permitting differential comparisons and validation of expressed mutations from limited and FFPE preserved samples, while reducing the data generation requirement. We conclude that cDNA hybrid capture has the potential to significantly improve transcriptome analysis from low-yield FFPE material. PMID:24814956

Cabanski, Christopher R; Magrini, Vincent; Griffith, Malachi; Griffith, Obi L; McGrath, Sean; Zhang, Jin; Walker, Jason; Ly, Amy; Demeter, Ryan; Fulton, Robert S; Pong, Winnie W; Gutmann, David H; Govindan, Ramaswamy; Mardis, Elaine R; Maher, Christopher A

2014-07-01

139

Quality of DNA extracted from saliva samples collected with the Oragene(TM) DNA self-collection kit  

PubMed Central

Background Large epidemiological studies in DNA biobanks have increasingly used less invasive methods for obtaining DNA samples, such as saliva collection. Although lower amounts of DNA are obtained as compared with blood collection, this method has been widely used because of its more simple logistics and increased response rate. The present study aimed to verify whether a storage time of 8?months decreases the quality of DNA from collected samples. Methods Saliva samples were collected with an OrageneTM DNA Self-Collection Kit from 4,110 subjects aged 14–15?years. The samples were processed in two aliquots with an 8-month interval between them. Quantitative and qualitative evaluations were carried out in 20% of the samples by spectrophotometry and genotyping. Descriptive analyses and paired t-tests were performed. Results The mean volume of saliva collected was 2.2?mL per subject, yielding on average 184.8??g DNA per kit. Most samples showed a Ratio of OD differences (RAT) between 1.6 and 1.8 in the qualitative evaluation. The evaluation of DNA quality by TaqMan®, High Resolution Melting (HRM), and restriction fragment length polymorphism-PCR (RFLP-PCR) showed a rate of success of up to 98% of the samples. The sample store time did not reduce either the quantity or quality of DNA extracted with the Oragene kit. Conclusion The study results showed that a storage period of 8?months at room temperature did not reduce the quality of the DNA obtained. In addition, the use of the Oragene kit during fieldwork in large population-based studies allows for DNA of high quantity and high quality.

2012-01-01

140

Relationship of DNA degradation by Saccharomyces cerevisiae Exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection  

PubMed Central

Homologous recombination is a major pathway for repair of DNA double-strand breaks. This repair process is initiated by resection of the 5?-terminated strand at the break site. In yeast, resection is carried out by three nucleolytic complexes: Mre11-Rad50-Xrs2, which functions at the initial step and also stimulates the two processive pathways, Sgs1-Dna2 and Exonuclease 1 (Exo1). Here we investigated the relationship between the three resection pathways with a focus on Exo1. Exo1 preferentially degrades the 5?-terminal stand of duplex DNA that is single stranded at the 3? end, in agreement with its role downstream of the Mre11-Rad50-Xrs2 complex. Replication protein A (RPA) stimulates DNA end resection by Exo1 by both preventing nonspecific binding of Exo1 to and preventing degradation of single-stranded DNA. Nucleolytic degradation of DNA by Exo1 is inhibited by the helicase-deficient Sgs1 K706A mutant protein and, reciprocally, the nuclease-deficient Exo1 D173A mutant protein inhibits DNA unwinding by Sgs1. Thus, the activities of Sgs1 and Exo1 at DNA ends are mutually exclusive, establishing biochemically that both machineries function independently in DNA end processing. We also reconstituted Sgs1-Top3-Rmi1-RPA-Dna2 and Exo1 resection reactions both individually and combined, either with or without the Mre11-Rad50-Xrs2 complex. We show that the yeast Sgs1-Dna2 and Exo1 pathways do not stimulate one another and function as independent and separate DNA end-processing machineries, even in the presence of the stimulatory Mre11-Rad50-Xrs2 complex.

Cannavo, Elda; Cejka, Petr; Kowalczykowski, Stephen C.

2013-01-01

141

Relationship of DNA degradation by Saccharomyces cerevisiae exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection.  

PubMed

Homologous recombination is a major pathway for repair of DNA double-strand breaks. This repair process is initiated by resection of the 5?-terminated strand at the break site. In yeast, resection is carried out by three nucleolytic complexes: Mre11-Rad50-Xrs2, which functions at the initial step and also stimulates the two processive pathways, Sgs1-Dna2 and Exonuclease 1 (Exo1). Here we investigated the relationship between the three resection pathways with a focus on Exo1. Exo1 preferentially degrades the 5?-terminal stand of duplex DNA that is single stranded at the 3? end, in agreement with its role downstream of the Mre11-Rad50-Xrs2 complex. Replication protein A (RPA) stimulates DNA end resection by Exo1 by both preventing nonspecific binding of Exo1 to and preventing degradation of single-stranded DNA. Nucleolytic degradation of DNA by Exo1 is inhibited by the helicase-deficient Sgs1 K706A mutant protein and, reciprocally, the nuclease-deficient Exo1 D173A mutant protein inhibits DNA unwinding by Sgs1. Thus, the activities of Sgs1 and Exo1 at DNA ends are mutually exclusive, establishing biochemically that both machineries function independently in DNA end processing. We also reconstituted Sgs1-Top3-Rmi1-RPA-Dna2 and Exo1 resection reactions both individually and combined, either with or without the Mre11-Rad50-Xrs2 complex. We show that the yeast Sgs1-Dna2 and Exo1 pathways do not stimulate one another and function as independent and separate DNA end-processing machineries, even in the presence of the stimulatory Mre11-Rad50-Xrs2 complex. PMID:23589858

Cannavo, Elda; Cejka, Petr; Kowalczykowski, Stephen C

2013-04-30

142

Mammalian Elongin A complex mediates DNA-damage-induced ubiquitylation and degradation of Rpb1  

PubMed Central

The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II (pol II) by suppressing transient pausing of the pol II at many sites along the DNA. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, which can form an isolable Elongin BC subcomplex. Here, we have shown that both the ubiquitylation and proteasomal degradation of the largest subunit of pol II (Rpb1) following UV-irradiation are significantly suppressed in Elongin A-deficient cells; however, in both cases suppression is rescued by transfection of wild-type Elongin A. Moreover, we have demonstrated that the Elongin A–Elongin BC complex is capable of assembling with the Cul5/Rbx2 module, and that this hetero-pentamer complex efficiently ubiquitylates Rpb1 in vitro. Mechanistic studies indicate that colocalization of Elongin A and Cul5 in cells and the interaction of Elongin A with the Ser5-phosphorylated form of Rpb1 are strongly enhanced following UV-irradiation. Taken together, our results suggest that mammalian Elongin A is directly involved in ubiquitylation and degradation of Rpb1 following DNA damage.

Yasukawa, Takashi; Kamura, Takumi; Kitajima, Shigetaka; Conaway, Ronald C; Conaway, Joan W; Aso, Teijiro

2008-01-01

143

Competence-Independent Activity of Pneumococcal Enda Mediates Degradation of Extracellular DNA and Nets and Is Important for Virulence  

PubMed Central

Membrane surface localized endonuclease EndA of the pulmonary pathogen Streptococcus pneumoniae (pneumococcus) is required for both genetic transformation and virulence. Pneumococcus expresses EndA during growth. However, it has been reported that EndA has no access to external DNA when pneumococcal cells are not competent for genetic transformation, and thus, unable to degrade extracellular DNA. Here, by using both biochemical and genetic methods, we demonstrate the existence of EndA-mediated nucleolytic activity independent of the competence state of pneumococcal cells. Pneumococcal mutants that are genetically deficient in competence development and genetic transformation have extracellular nuclease activity comparable to their parental wild type, including their ability to degrade neutrophil extracellular traps (NETs). The autolysis deficient ?lytA mutant and its isogenic choline-treated parental wild-type strain D39 degrade extracellular DNA readily, suggesting that partial cell autolysis is not required for DNA degradation. We show that EndA molecules are secreted into the culture medium during the growth of pneumococcal cells, and contribute substantially to competence-independent nucleolytic activity. The competence-independent activity of EndA is responsible for the rapid degradation of DNA and NETs, and is required for the full virulence of Streptococcus pneumoniae during lung infection.

Zhu, Luchang; Kuang, Zhizhou; Wilson, Brenda A.; Lau, Gee W.

2013-01-01

144

An empirical test of DNA mark–recapture sampling strategies for grizzly bears  

Microsoft Academic Search

Despite the widespread use of DNA mark-recapture for estimation of grizzly bear (Ursus arctos) population size, there have been no designed experiments of DNA sampling strategies. We designed a large-scale study (8,820 km2) in the foothills of Alberta, Canada, to test sampling strategies associated with the hair snag DNA method. The main sampling method for this project used a traditional

John Boulanger; Michael Proctor; Stefan Himmer; Gordon Stenhouse; David Paetkau; Jerome Cranston

2006-01-01

145

A Yeast GSK-3 Kinase Mck1 Promotes Cdc6 Degradation to Inhibit DNA Re-Replication  

PubMed Central

Cdc6p is an essential component of the pre-replicative complex (pre-RC), which binds to DNA replication origins to promote initiation of DNA replication. Only once per cell cycle does DNA replication take place. After initiation, the pre-RC components are disassembled in order to prevent re-replication. It has been shown that the N-terminal region of Cdc6p is targeted for degradation after phosphorylation by Cyclin Dependent Kinase (CDK). Here we show that Mck1p, a yeast homologue of GSK-3 kinase, is also required for Cdc6 degradation through a distinct mechanism. Cdc6 is an unstable protein and is accumulated in the nucleus only during G1 and early S-phase in wild-type cells. In mck1 deletion cells, CDC6p is stabilized and accumulates in the nucleus even in late S phase and mitosis. Overexpression of Mck1p induces rapid Cdc6p degradation in a manner dependent on Threonine-368, a GSK-3 phosphorylation consensus site, and SCFCDC4. We show evidence that Mck1p-dependent degradation of Cdc6 is required for prevention of DNA re-replication. Loss of Mck1 activity results in synthetic lethality with other pre-RC mutants previously implicated in re-replication control, and these double mutant strains over-replicate DNA within a single cell cycle. These results suggest that a GSK3 family protein plays an unexpected role in preventing DNA over-replication through Cdc6 degradation in Saccharomyces cerevisiae. We propose that both CDK and Mck1 kinases are required for Cdc6 degradation to ensure a tight control of DNA replication.

Ikui, Amy E.; Rossio, Valentina; Schroeder, Lea; Yoshida, Satoshi

2012-01-01

146

A yeast GSK-3 kinase Mck1 promotes Cdc6 degradation to inhibit DNA re-replication.  

PubMed

Cdc6p is an essential component of the pre-replicative complex (pre-RC), which binds to DNA replication origins to promote initiation of DNA replication. Only once per cell cycle does DNA replication take place. After initiation, the pre-RC components are disassembled in order to prevent re-replication. It has been shown that the N-terminal region of Cdc6p is targeted for degradation after phosphorylation by Cyclin Dependent Kinase (CDK). Here we show that Mck1p, a yeast homologue of GSK-3 kinase, is also required for Cdc6 degradation through a distinct mechanism. Cdc6 is an unstable protein and is accumulated in the nucleus only during G1 and early S-phase in wild-type cells. In mck1 deletion cells, CDC6p is stabilized and accumulates in the nucleus even in late S phase and mitosis. Overexpression of Mck1p induces rapid Cdc6p degradation in a manner dependent on Threonine-368, a GSK-3 phosphorylation consensus site, and SCF(CDC4). We show evidence that Mck1p-dependent degradation of Cdc6 is required for prevention of DNA re-replication. Loss of Mck1 activity results in synthetic lethality with other pre-RC mutants previously implicated in re-replication control, and these double mutant strains over-replicate DNA within a single cell cycle. These results suggest that a GSK3 family protein plays an unexpected role in preventing DNA over-replication through Cdc6 degradation in Saccharomyces cerevisiae. We propose that both CDK and Mck1 kinases are required for Cdc6 degradation to ensure a tight control of DNA replication. PMID:23236290

Ikui, Amy E; Rossio, Valentina; Schroeder, Lea; Yoshida, Satoshi

2012-01-01

147

An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis  

Microsoft Academic Search

A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonucleasePalI. The extraction method was used to obtain DNA from a

L. Arlene Porteous; John L. Armstrong; Ramon J. Seidler; Lidia S. Watrud

1994-01-01

148

Whole genome amplification: abundant supplies of DNA from precious samples or clinical specimens  

Microsoft Academic Search

Whole genome amplification methods are used to generate the large amounts of DNA that are required for genetic testing. Preparation of genomic DNA from clinical samples is a bottleneck in high-throughput genotyping and is frequently limited by the amount of specimen available. Precious DNA collections used for association and linkage analysis can be a nonrenewable resource available to only a

Roger S. Lasken; Michael Egholm

2003-01-01

149

Detection of polycyclic aromatic hydrocarbon degradation genes in different soil bacteria by polymerase chain reaction and DNA hybridization  

Microsoft Academic Search

Twenty different strains of Pseudomonas, Mycobacterium, Gordona, Sphingomonas, Rhodococcus and Xanthomonas which degrade polycyclic aromatic hydrocarbons (PAH) were characterized in respect to genes encoding degradation enzymes for PAH. Genomic DNA from these strains was hybridized with a fragment of ndoB, coding for the large iron sulfur protein (ISP?) of the naphthalene dioxygenase from Pseudomonas putida PaW736 (NCIB 9816). A group

Christian Hamann; Jörg Hegemann; Armin Hildebrandt

1999-01-01

150

Physical degradation of genomic DNA of soybean flours does not impair relative quantification of its transgenic content  

Microsoft Academic Search

In this paper, four different physical treatments (microwaves, heating by conduction, sonication and pressure autoclaving)\\u000a were performed to degrade a pure DNA extract, and their influence on GMO quantification was studied. The aim was to check\\u000a the hypothesis that processing of agrofood products results in a similar degradation rate for both the transgenic target and\\u000a the specific target. Indeed we

Frédéric Debode; Eric Janssen; Gilbert Berben

2007-01-01

151

A Simple Method of Genomic DNA Extraction from Human Samples for PCR-RFLP Analysis  

PubMed Central

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4–6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.

Ghatak, Souvik; Muthukumaran, Rajendra Bose; Nachimuthu, Senthil Kumar

2013-01-01

152

A simple method of genomic DNA extraction from human samples for PCR-RFLP analysis.  

PubMed

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4-6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability. PMID:24294115

Ghatak, Souvik; Muthukumaran, Rajendra Bose; Nachimuthu, Senthil Kumar

2013-12-01

153

A simple DNA extraction method for marijuana samples used in amplified fragment length polymorphism (AFLP) analysis.  

PubMed

As a first step in developing a molecular method for the individualization of marijuana samples, we evaluated a plant DNA extraction kit. The QIAGEN plant DNeasy method uses a spin column format for recovery of DNA and is effective for obtaining high molecular weight DNA from leaf, flower (bud), and seed samples of marijuana. The average DNA yield was 125-500 ng per 100 milligrams of fresh plant tissue. The recovered DNA was of polymerase chain reaction (PCR) quality as measured by the ability to generate reproducible amplified fragment length polymorphism (AFLP) profiles. AFLP is a technique used to create a DNA profile for plant varieties and is being applied to marijuana samples by the authors to link growers and distributors of clonal material. The QIAGEN plant DNeasy method was simple, efficient, and reproducible for processing small quantities of marijuana into DNA. PMID:12664992

Miller Coyle, Heather; Shutler, Gary; Abrams, Sharon; Hanniman, Janet; Neylon, Suzanne; Ladd, Carll; Palmbach, Timothy; Lee, Henry C

2003-03-01

154

DNA Isolation from Small Tissue Samples Using Salt and Spermine.  

National Technical Information Service (NTIS)

Common DNA isolation methods rely upon protein denaturation by organic solvents such as phenol and chloroform. These solvents pose some risk to the user and require special disposal procedures. The authors have previously reported a method for isolating D...

J. A. Ross G. B. Nelson K. L. Holden

1991-01-01

155

Degradation of DNA by iron-bleomycin: mechanistic implications of product /sup 18/O incorporation  

SciTech Connect

Interaction of d(CGCGCG) with Bleomycin (BLM), activated either with Fe(III) and H/sub 2/O/sub 2/ or Fe(II), O/sub 2/ and one electron, results in production of cytosine and a modified oligonucleotide strand (1). Reduction of 1 with NaBD/sub 4/ followed by enzymatic digestion, derivatization, and GC-MS permits the identification of 2-deoxypentitols-1,4-d/sub 2/ as their tetra-trimethylsilyl (TMS) derivatives. Similar products have also been isolated from calf thymus DNA and poly(dG-dC). These results provide unequivocal evidence for the intermediacy of a 4' ketone, 1' aldehyde modified carbohydrate. An alternate mode of DNA degradation requires additional O/sub 2/ and leads to formation of 3' phosphoglycolate termini and base propenals. Glycolate (GA), released from calf thymus DNA, poly(dA-dT) or d(CGCGCG) by enzymatic digestion, can be isolated by chromatography on DEAE Sephadex, silylated and analyzed by GC-MS. This analysis, after incubation with Fe(II) x /sup 18/O/sub 2/ x BLM or Fe(III) x H/sub 2/ /sup 16/O/sub 2/ x BLM plus /sup 18/O/sub 2/, reveals the incorporation of a single atom of /sup 18/O at the C-1 position. Pulse-chase experiments demonstrate that it is the excess molecular oxygen and not the O/sub 2/ required for drug activation that is incorporated into the carboxylate group of 3' phosphoglycolate and provide evidence for the proposed addition of O/sub 2/ to a C4' carbon radical. Isotopic enrichments of the other products of DNA oxidation, formed in the presence of /sup 18/O/sub 2/ are also being determined.

Rabow, L.E.; McGall, G.H.; Stubbe, J.; Kozarich, J.W.

1987-05-01

156

Comparison of KRAS Mutation Assessment in Tumor DNA and Circulating Free DNA in Plasma and Serum Samples  

PubMed Central

Testing for mutations in the KRAS oncogene for patients with metastatic colorectal cancer (mCRC) is generally performed using DNA from formalin-fixed paraffin-embedded tumor tissue; however, access to specimens can be limited and analysis challenging. This study assessed the identification of KRAS mutations in circulating free DNA (cfDNA) using a commercially available KRAS polymerase chain reaction (PCR) kit. Matched plasma, serum and tumor samples were available from 71 patients with mCRC who had received prior therapy but whose disease progressed following therapy. Yields of cfDNA from plasma and serum samples were comparable. Analyses were successful in 70/71 plasma-extracted samples (specificity: 97%, sensitivity: 31%) and 67/71 serum- extracted samples (specificity: 100%, sensitivity: 25%). This study demonstrates that KRAS mutations can be detected in cfDNA using a commercially available KRAS PCR kit, confirming cfDNA as a potential alternative source of tumor DNA in a diagnostic setting if access to archival tumor specimens is limited.

Morgan, Shethah R.; Whiteley, Jessica; Donald, Emma; Smith, John; Eisenberg, Marcia T.; Kallam, Eddie; Kam-Morgan, Lauren

2012-01-01

157

Analysis of atrazine-degrading microbial communities in soils using most-probable-number enumeration, DNA hybridization, and inhibitors  

Microsoft Academic Search

The purpose of this study was to determine whether there was an association between kinetics of atrazine mineralization, the number and type of atrazine-degrading microorganisms, and the presence of three genes representing different steps in the degradative pathway of atrazine in three soils with different histories of atrazine application. Composite soil samples were collected from two agricultural fields and one

Ellen B Ostrofsky; Jayne B Robinson; Samuel J Traina; Olli H Tuovinen

2002-01-01

158

Multiple pathways are involved in DNA degradation during keratinocyte terminal differentiation.  

PubMed

Loss of the nucleus is a critical step in keratinocyte terminal differentiation. To elucidate the mechanisms involved, we focused on two characteristic events: nuclear translocation of N-terminal fragment of profilaggrin and caspase-14-dependent degradation of the inhibitor of caspase-activated DNase (ICAD). First, we demonstrated that epidermal mesotrypsin liberated a 55-kDa N-terminal fragment of profilaggrin (FLG-N) and FLG-N was translocated into the nucleus. Interestingly, these cells became TUNEL positive. Mutation in the mesotrypsin-susceptible Arg-rich region between FLG-N and the first filaggrin domain abolished these changes. Furthermore, caspase-14 caused limited proteolysis of ICAD, followed by accumulation of caspase-activated DNase (CAD) in TUNEL-positive nuclei. Knockdown of both proteases resulted in a significant increase of remnant nuclei in a skin equivalent model. Immunohistochemical study revealed that both caspase-14 and mesotrypsin were markedly downregulated in parakeratotic areas of lesional skin from patients with atopic dermatitis and psoriasis. Collectively, our results indicate that at least two pathways are involved in the DNA degradation process during keratinocyte terminal differentiation. PMID:24743736

Yamamoto-Tanaka, M; Makino, T; Motoyama, A; Miyai, M; Tsuboi, R; Hibino, T

2014-01-01

159

Estimating occupancy and abundance of stream amphibians using environmental DNA from filtered water samples  

USGS Publications Warehouse

Environmental DNA (eDNA) methods for detecting aquatic species are advancing rapidly, but with little evaluation of field protocols or precision of resulting estimates. We compared sampling results from traditional field methods with eDNA methods for two amphibians in 13 streams in central Idaho, USA. We also evaluated three water collection protocols and the influence of sampling location, time of day, and distance from animals on eDNA concentration in the water. We found no difference in detection or amount of eDNA among water collection protocols. eDNA methods had slightly higher detection rates than traditional field methods, particularly when species occurred at low densities. eDNA concentration was positively related to field-measured density, biomass, and proportion of transects occupied. Precision of eDNA-based abundance estimates increased with the amount of eDNA in the water and the number of replicate subsamples collected. eDNA concentration did not vary significantly with sample location in the stream, time of day, or distance downstream from animals. Our results further advance the implementation of eDNA methods for monitoring aquatic vertebrates in stream habitats.

Pilliod, David S.; Goldberg, Caren S.; Arkle, Robert S.; Waits, Lisette P.

2013-01-01

160

A two-step electrodialysis method for DNA purification from polluted metallic environmental samples.  

PubMed

Extracting DNA from samples of polluted environments using standard methods often results in low yields of poor-quality material unsuited to subsequent manipulation and analysis by molecular biological techniques. Here, we report a novel two-step electrodialysis-based method for the extraction of DNA from environmental samples. This technique permits the rapid and efficient isolation of high-quality DNA based on its acidic nature, and without the requirement for phenol-chloroform-isoamyl alcohol cleanup and ethanol precipitation steps. Subsequent PCR, endonuclease restriction, and cloning reactions were successfully performed utilizing DNA obtained by electrodialysis, whereas some or all of these techniques failed using DNA extracted with two alternative methods. We also show that his technique is applicable to purify DNA from a range of polluted and nonpolluted samples. PMID:18601228

Rodríguez-Mejía, José Luis; Martínez-Anaya, Claudia; Folch-Mallol, Jorge Luis; Dantán-González, Edgar

2008-08-01

161

Sources of pre-analytical variations in yield of DNA extracted from blood samples: analysis of 50,000 DNA samples in EPIC.  

PubMed

The European Prospective Investigation into Cancer and nutrition (EPIC) is a long-term, multi-centric prospective study in Europe investigating the relationships between cancer and nutrition. This study has served as a basis for a number of Genome-Wide Association Studies (GWAS) and other types of genetic analyses. Over a period of 5 years, 52,256 EPIC DNA samples have been extracted using an automated DNA extraction platform. Here we have evaluated the pre-analytical factors affecting DNA yield, including anthropometric, epidemiological and technical factors such as center of subject recruitment, age, gender, body-mass index, disease case or control status, tobacco consumption, number of aliquots of buffy coat used for DNA extraction, extraction machine or procedure, DNA quantification method, degree of haemolysis and variations in the timing of sample processing. We show that the largest significant variations in DNA yield were observed with degree of haemolysis and with center of subject recruitment. Age, gender, body-mass index, cancer case or control status and tobacco consumption also significantly impacted DNA yield. Feedback from laboratories which have analyzed DNA with different SNP genotyping technologies demonstrate that the vast majority of samples (approximately 88%) performed adequately in different types of assays. To our knowledge this study is the largest to date to evaluate the sources of pre-analytical variations in DNA extracted from peripheral leucocytes. The results provide a strong evidence-based rationale for standardized recommendations on blood collection and processing protocols for large-scale genetic studies. PMID:22808065

Caboux, Elodie; Lallemand, Christophe; Ferro, Gilles; Hémon, Bertrand; Mendy, Maimuna; Biessy, Carine; Sims, Matt; Wareham, Nick; Britten, Abigail; Boland, Anne; Hutchinson, Amy; Siddiq, Afshan; Vineis, Paolo; Riboli, Elio; Romieu, Isabelle; Rinaldi, Sabina; Gunter, Marc J; Peeters, Petra H M; van der Schouw, Yvonne T; Travis, Ruth; Bueno-de-Mesquita, H Bas; Canzian, Federico; Sánchez, Maria-José; Skeie, Guri; Olsen, Karina Standahl; Lund, Eiliv; Bilbao, Roberto; Sala, Núria; Barricarte, Aurelio; Palli, Domenico; Navarro, Carmen; Panico, Salvatore; Redondo, Maria Luisa; Polidoro, Silvia; Dossus, Laure; Boutron-Ruault, Marie Christine; Clavel-Chapelon, Françoise; Trichopoulou, Antonia; Trichopoulos, Dimitrios; Lagiou, Pagona; Boeing, Heiner; Fisher, Eva; Tumino, Rosario; Agnoli, Claudia; Hainaut, Pierre

2012-01-01

162

DNA Profiling of Convicted Offender Samples for the Combined DNA Index System  

ERIC Educational Resources Information Center

The cornerstone of forensic chemistry is that a perpetrator inevitably leaves trace evidence at a crime scene. One important type of evidence is DNA, which has been instrumental in both the implication and exoneration of thousands of suspects in a wide range of crimes. The Combined DNA Index System (CODIS), a network of DNA databases, provides…

Millard, Julie T

2011-01-01

163

Current sample handling methods for measurement of platinum-DNA adducts in leucocytes in man lead to discrepant results in DNA adduct levels and DNA repair.  

PubMed Central

DNA adduct levels were measured with atomic spectroscopy in white blood cells (WBCs) from patients with solid tumours who were treated with six weekly courses of cisplatin. In 21 patients (I) the WBCs were collected after thawing frozen whole-blood samples according to a previously described method. In 32 other patients (II) WBCs were collected immediately after blood sample collection. The two methods for WBC collection were also compared in vitro. The maximal DNA adduct levels in vivo after the first course were in I 2.48 +/- 1.14 and in II 1.28 +/- 0.40 pg of platinum per microgram of DNA (P < 0.0001). The DNA 'repair' in the first course (DNA adduct level at the end of the infusion minus the level 15 h post infusion) was in I 40% +/- 29% and in II 18% +/- 29% (P = 0.009). These differences were consistent in all measured courses. In vitro, the DNA adduct levels in the freshly prepared WBCs were significantly lower at 0, 1 and 4, but not 24 h, after start of the incubation with cisplatin than in the WBCs collected after freezing and thawing the blood sample. The same experiment with carboplatin in vitro also resulted in significantly lower adducts in freshly isolated WBCs. The higher DNA adduct levels and DNA 'repair' in I are caused by remaining unbound cisplatin in the sample tubes, which can form DNA adducts ex vivo. The same results in vivo can be anticipated when carboplatin is used.

Ma, J.; Verweij, J.; Planting, A. S.; de Boer-Dennert, M.; van Ingen, H. E.; van der Burg, M. E.; Stoter, G.; Schellens, J. H.

1995-01-01

164

The ribonucleotide reductase inhibitor, Sml1, is sequentially phosphorylated, ubiquitylated and degraded in response to DNA damage.  

PubMed

Regulation of ribonucleotide reductase (RNR) is important for cell survival and genome integrity in the face of genotoxic stress. The Mec1/Rad53/Dun1 DNA damage response kinase cascade exhibits multifaceted controls over RNR activity including the regulation of the RNR inhibitor, Sml1. After DNA damage, Sml1 is degraded leading to the up-regulation of dNTP pools by RNR. Here, we probe the requirements for Sml1 degradation and identify several sites required for in vivo phosphorylation and degradation of Sml1 in response to DNA damage. Further, in a strain containing a mutation in Rnr1, rnr1-W688G, mutation of these sites in Sml1 causes lethality. Degradation of Sml1 is dependent on the 26S proteasome. We also show that degradation of phosphorylated Sml1 is dependent on the E2 ubiquitin-conjugating enzyme, Rad6, the E3 ubiquitin ligase, Ubr2, and the E2/E3-interacting protein, Mub1, which form a complex previously only implicated in the ubiquitylation of Rpn4. PMID:20566477

Andreson, Bethany L; Gupta, Amitabha; Georgieva, Bilyana P; Rothstein, Rodney

2010-10-01

165

Optimal selection strategies for QTL mapping using pooled DNA samples  

Microsoft Academic Search

The cost of large-scale association studies may be reduced substantially by analysis of pooled DNA from multiple individuals. Here we examine the optimal symmetric and asymmetric designs for pooling experiments for quantitative traits under a range of assumptions about the underlying genetic model and the sources of experimental errors in allele frequency estimation. The results indicate that, in the absence

Ansar Jawaid; Joel S Bader; Shaun Purcell; Stacey S Cherny; Pak Sham

2002-01-01

166

Effect of ascorbic acid and curcumin on quercetin-induced nuclear DNA damage, lipid peroxidation and protein degradation.  

PubMed

The effects of ascorbic acid and curcumin on quercetin-induced DNA damage, lipid peroxidation protein degradation were investigated in a model system of isolated rat-liver nuclei under aerobic conditions and in the presence of equimolar concentrations of iron or copper. Neither ascorbic acid nor curcumin inhibited quercetin-induced nuclear DNA damage, lipid peroxidation, or protein degradation. In fact, both antioxidants stimulated the oxidative damage to nuclear macromolecules. Ascorbic acid significantly increased the quercetin-induced nuclear DNA damage in the presence of either iron or copper. The increases in quercetin-induced nuclear lipid peroxidation and protein degradation by ascorbic acid were statistically significant only in the presence of iron or copper, respectively. Similarly, stimulation of quercetin-induced DNA damage and lipid peroxidation by curcumin was statistically significant only in the presence of copper or iron, respectively. Curcumin had no significant effect on nuclear protein degradation. These results demonstrate the pro-oxidant properties of ascorbic acid and curcumin, compounds that also demonstrate antioxidant and anticarcinogenic properties. Ascorbic acid and curcumin may therefore each have a dual role in carcinogenesis. PMID:1576592

Sahu, S C; Washington, M C

1992-04-30

167

Molecular Diagnosis of Strongyloides stercoralis Infection by PCR Detection of Specific DNA in Human Stool Samples  

PubMed Central

Background Strongyloidiasis is mostly an asymptomatic infection and diagnosis of latent infections is difficult due to limitations of current parasitological and serological methods. This study was conducted to set up a PCR-based method for molecular diagnosis of Strongyloides stercoralis infection by detection of copro-DNA in stool samples. Methods A total of 782 fresh stool samples were collected and examined by agar plate culture. Among those sixteen stool samples, which confirmed to be infected with S. stercoralis were examined as positive control to set up each single and nested PCR, using two primer sets designing to amplify partial ribosomal DNA of S. stercoralis genome. Since, single PCR method yielded higher efficacy in detecting positive samples, in the second step, 30 stool samples, which found negative for S. stercoralis by agar plate culture of single stool sample, were examined by single PCR. Data analysis was performed using McNemar's ?2 test, with consideration of a P-value of <0.05 as indication of significant difference. Results In amplification of DNA extracted from stool samples, single PCR detected S. stercoralis DNA target in all 16 positive samples, while nested PCR amplified DNA in only 75% of samples. In the second step, single PCR amplified S. stercoralis extracted DNA in 5 out of 30 samples which were negative by coproculture. Conclusion Single PCR method amplifying a short (100bp) target represented more efficacies for detection of S. stercoralis in faecal examination compared to agar plate culture and nested PCR, which amplified longer target.

Moghaddassani, H; Mirhendi, H; Hosseini, M; Rokni, MB; Mowlavi, Gh; Kia, Eb

2011-01-01

168

A novel method to realize the transition from silver nanowires to nanoplates based on the degradation of DNA  

Microsoft Academic Search

Silver “nano-necklaces” and nanoplates in DNA\\/Tris–EDTA (TE) solution are prepared using hydrothermal method. The nano-necklaces\\u000a are composed of many spherical silver nanoparticles which are joined together by the DNA chain. Further the transition from\\u000a silver nano-necklaces to triangular and hexagonal nanoplates is realized based on the degradation of DNA. Transmission electron\\u000a microscopy, selected area electron diffraction, ultraviolet–visible spectroscopy, X-ray diffraction,

Chuanhao Li; Yuhua Shen; Anjian Xie; Juan Wang; Qingfeng Zhang; Shikuo Li

2010-01-01

169

Identification of a reactive degradation zone at a landfill leachate plume fringe using high resolution sampling and incubation techniques.  

PubMed

Vertical small-scale variation in phenoxy acid herbicide degradation across a landfill leachate plume fringe was studied using laboratory degradation experiments. Sediment cores (subdivided into 5 cm segments) were collected in the aquifer and the sediment and porewater were used for microcosm experiments (50 experiments) and for determination of solid organic carbon, solid-water partitioning coefficients, specific phenoxy acid degraders and porewater chemistry. Results from a multi-level sampler installed next to the cores provided information on the plume position and oxygen concentration in the groundwater. Oxygen concentration was controlled individually in each microcosm to mimic the conditions at their corresponding depths. A highly increased degradation potential existed at the narrow plume fringe (37.7 to 38.6 masl), governed by the presence of phenoxy acids and oxygen. This resulted in the proliferation of a microbial population of specific phenoxy acid degraders, which further enhanced the degradation potential for phenoxy acids at the fringe. The results illustrate the importance of fringe degradation processes in contaminant plumes. Furthermore, they highlight the relevance of using high-resolution sampling techniques as well as controlled microcosm experiments in the assessment of the natural attenuation capacity of contaminant plumes in groundwater. PMID:16524640

Tuxen, Nina; Albrechtsen, Hans-Jørgen; Bjerg, Poul L

2006-05-30

170

A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples.  

PubMed

Differences in the composition of the gut microbiota have been associated with a range of diseases using culture-independent methods. Reliable extraction of nucleic acid is a key step in identifying the composition of the faecal microbiota. Five widely used commercial deoxyribonucleic acid (DNA) extraction kits (QIAsymphony® Virus/Bacteria Midi Kit (kit QS), ZR Fecal DNA MiniPrep™ (kit Z), QIAamp® DNA Stool Mini Kit (kit QA), Ultraclean® Fecal DNA Isolation Kit (kit U) and PowerSoil® DNA Isolation Kit (kit P)) were evaluated, using human faecal samples. Yield, purity and integrity of total genomic DNA were compared spectrophotometrically and using gel electrophoresis. Three bacteria, commonly found in human faeces were quantified using real time polymerase chain reaction (qPCR) and total bacterial diversity was studied using denaturing gradient gel electrophoresis (DGGE) as well as terminal restriction fragment length polymorphism (T-RFLP). The measurements of DNA yield and purity exhibited variations between the five kits tested in this study. Automated kit QS exhibited the best quality and highest quantity of DNA. All kits were shown to be reproducible with CV values?0.46 for DNA extraction. qPCR results showed that all kits were uniformly efficient for extracting DNA from the selected target bacteria. DGGE and T-RFLP produced the highest diversity scores for DNA extracted using kit Z (H'=2.30 and 1.27) and kit QS (H'=2.16 and 0.94), which also extracted the highest DNA yields compared to the other kits assessed. PMID:23684993

Claassen, Shantelle; du Toit, Elloise; Kaba, Mamadou; Moodley, Clinton; Zar, Heather J; Nicol, Mark P

2013-08-01

171

PNA microarrays for hybridisation of unlabelled DNA samples  

Microsoft Academic Search

Several strategies have been developed for the pro- duction of peptide nucleic acid (PNA) microarrays by parallel probe synthesis and selective coupling of full-length molecules. Such microarrays were used for direct detection of the hybridisation of unlabelled DNA by time-of-flight secondary ion mass spectrometry. PNAs were synthesised by an automated process on filter-bottom microtitre plates. The resulting molecules were released

Ole Brandt; Julia Feldner; Achim Stephan; Markus Schroder; Martina Schnolzer; Heinrich F. Arlinghaus; D. Hoheisel; Anette Jacob

2003-01-01

172

Comparison of five commercial DNA extraction kits for the recovery of Francisella tularensis DNA from spiked soil samples.  

PubMed

Francisella tularensis is the etiologic agent of the zoonotic disease tularemia and is thought to be maintained in the environment principally by various terrestrial and aquatic vertebrate animals. The organism is known to persist in water or mud for long periods of time and Francisella-specific DNA has been identified from water and soil. To gain a better understanding of the ecology and epidemiology of F. tularensis, it will be important to further explore its distribution in the environment. Therefore, methods must be established to efficiently extract Francisella-specific DNA from the soil and be able to eliminate potential PCR inhibitors. Thus, we evaluated five commercial DNA extraction kits for their ability to recover F. tularensis-specific DNA from soil samples and eliminate potential PCR inhibitors. The kits evaluated included the Puregene DNA purification kit, QIAamp Stool Mini kit, Epicentre Biotech SoilMaster DNA extraction kit, and the UltraClean and PowerMax soil DNA isolation kits from MoBio. Soil samples were spiked with gamma-irradiated F. tularensis SHU-4 strain (corresponding to a range from 10 to 10(5)CFU). Spiked samples were extracted with each kit and evaluated using a F. tularensis-specific real-time PCR assay and an internal positive control assay that measures the presence of potential PCR inhibitors. DNA extraction using the UltraClean and PowerMax kits resulted in the most consistently positive results at the lowest limit of detection (20 and 100CFU/g soil, respectively) for all soil types tested, suggesting that these kits can provide the most sensitive methods for extracting F. tularensis from environmental soil samples. Processing time and cost were also evaluated. PMID:17011748

Whitehouse, Chris A; Hottel, Hannah E

2007-04-01

173

Electrochemical DNA biosensor as a screening tool for the detection of toxicants in water and wastewater samples  

Microsoft Academic Search

Applications of a disposable electrochemical DNA biosensor to standard solutions and to real samples are reported. The DNA biosensor is assembled by immobilising the double stranded calf thymus DNA on the surface of a disposable carbon screen-printed electrode. The immobilised ds-DNA interacts with the sample for 2 min; then is washed and immersed in a clean buffer where the analytical

F. Lucarelli; I. Palchetti; G. Marrazza; M. Mascini

2002-01-01

174

Isolation of DNA from bacterial samples of the human gastrointestinal tract  

Microsoft Academic Search

The human gastrointestinal (GI) tract contains a complex microbial community that develops in time and space. The most widely used approaches to study microbial diversity and activity are all based on the analysis of nucleic acids, DNA, rRNA and mRNA. Here, we present a DNA isolation protocol that is suitable for a wide variety of GI tract samples, including biopsies

Erwin G Zoetendal; Hans GHJ Heilig; Eline S Klaassens; Carien CGM Booijink; Michiel Kleerebezem; Hauke Smidt; Willem M de Vos

2006-01-01

175

A disposable DNA sample preparation microfluidic chip for nucleic acid probe assay  

Microsoft Academic Search

A new DNA sample preparation microfluidic chip for Nucleic Acid (NA) probe assay has been proposed. The proposed microfluidic chip is composed of three parts: microfilter, micromixer and DNA purification chip. We have fabricated a microsieve type filter with an array of 2.2 ?m diameter holes. We have also demonstrated the mixing can be successfully achieved for low Reynolds number

Joon-Ho Kim; Byoung-Gyun Kim; Hyukjun Nam; Dae-Eun Park; Kwang-Seok Yun; Jun-Bo Yoon; Jichang You; Euisik Yoon

2002-01-01

176

Multiplex SNP genotyping in pooled DNA samples by a four-colour microarray system  

Microsoft Academic Search

We selected 125 candidate single nucleotide poly- morphisms (SNPs) in genes belonging to the human type 1 interferon (IFN) gene family and the genes coding for proteins in the main type 1 IFN signalling pathway by screening databases and by in silico comparison of DNA sequences. Using quantitative analysis of pooled DNA samples by solid-phase mini-sequencing, we found that only

Katarina Lindroos; Snaevar Sigurdsson; Karin Johansson; Lars Ronnblom; Ann-Christine Syvanen

2002-01-01

177

Sample processing for DNA chip array-based analysis of enterohemorrhagic Escherichia coli (EHEC)  

Microsoft Academic Search

BACKGROUND: Exploitation of DNA-based analyses of microbial pathogens, and especially simultaneous typing of several virulence-related genes in bacteria is becoming an important objective of public health these days. RESULTS: A procedure for sample processing for a confirmative analysis of enterohemorrhagic Escherichia coli (EHEC) on a single colony with DNA chip array was developed and is reported here. The protocol includes

Pascal Basselet; Grzegorz Wegrzyn; Sven-Olof Enfors; Magdalena Gabig-Ciminska

2008-01-01

178

Non-lethal sampling of honey bee, Apis mellifera , DNA using wing tips  

Microsoft Academic Search

DNA sampling of insects frequently relies upon lethal or invasive methods. Because insect colonies contain numerous workers it is often possible to destructively sample workers for genetic analysis. However, this is not possible if queens or workers must remain alive after sampling. Neither is it possible to remove an entire leg, wing or other appendage as this will often hinder

Nicolas Châline; Francis L. W. Ratnieks; Nigel E. Raine; Nichola S. Badcock; Terry Burke

2004-01-01

179

DNA by Mail: An Inexpensive and Noninvasive Method for Collecting DNA Samples from Widely Dispersed Populations  

Microsoft Academic Search

As specific genes are identified that are associated with behavior, it becomes increasingly important for behavioral geneticists to be able to incorporate these genes in their research. Rather than using blood, DNA can be extracted from cheek swabs, which makes it possible to obtain DNA inexpensively by mail from large, widely dispersed individuals. The purpose of this paper is to

Bernard Freeman; John Powell; David Ball; Linzy Hill; Ian Craig; Robert Plomin

1997-01-01

180

Mitochondrial DNA control region variation in a Kuwaiti population sample.  

PubMed

Mitochondrial control region (16024-576) sequences were generated from 381 Kuwaiti samples. Previously, these samples were typed with the AmpF?STR(®) Identifiler(®) kit (Applied Biosystems, Foster City, California). Automated high throughput lab processing combined with a redundant sequencing strategy and multiple reviews of the raw electropherograms ensure the high quality of these sequences and their utility as reference population data for Kuwait. PMID:21555259

Scheible, Melissa; Alenizi, Mohammad; Sturk-Andreaggi, Kimberly; Coble, Michael D; Ismael, Samar; Irwin, Jodi A

2011-08-01

181

Optimised sample preparation of synovial fluid for detection of Chlamydia trachomatis DNA by polymerase chain reaction  

PubMed Central

OBJECTIVE—To optimise sample preparation of synovial fluid for Chlamydia trachomatis (CT) specific polymerase chain reaction (PCR).?METHODS—Serial dilutions of purified CT elementary bodies in synovial fluid were prepared. The synovial fluid pellet was processed by eight different methods of sample preparation. Then samples were analysed by CT specific PCR. The sensitivity of PCR was the basis of ranking of the eight different methods.?RESULTS—Highest sensitivity was achieved by methods including an additional step of DNA isolation. Additional extraction of protein and polysaccharides by cetyltrimethylammonium bromide (CTAB) increased sensitivity. Addition of hyaluronidase did not increase sensitivity of QIAEX-DNA extraction but was necessary, however, before phenol-chloroform-DNA extraction.?CONCLUSIONS—The method of synovial fluid sample preparation significantly influences the sensitivity of subsequent PCR. Additional DNA isolation and extraction of PCR inhibitors by CTAB led to higher sensitivity.?? Keywords: Chlamydia trachomatis; polymerase chain reaction; synovial fluid

Kuipers, J.; Nietfeld, L.; Dreses-Werringloe..., U.; Koehler, L.; Wollenhaupt, J.; Zeidler, H.; Hammer, M.

1999-01-01

182

DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.  

PubMed

Accelerator mass spectrometry (AMS) is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels. PMID:24623226

Dingley, Karen H; Ubick, Esther A; Vogel, John S; Ognibene, Ted J; Malfatti, Michael A; Kulp, Kristen; Haack, Kurt W

2014-01-01

183

PEG-assisted DNA solubilization in organic solvents for preparing cytosol specifically degradable PEG/DNA nanogels.  

PubMed

DNA was dissolved in selected organic solvents in the presence of poly(ethylene glycol) (PEG). Nanoscale PEG/DNA complex (approximately 100 nm) was produced in dimethylsulfoxide (DMSO) phase. Using a thiol-functionalized six-arm branched PEG for DNA solubilization, the PEG/DNA nanocomplex was cross-linked through the formation of disulfide linkages between the thiol groups, resulting in the production of stable PEG/DNA nanogels in aqueous solution. DNA release from the nanogels could be modulated by changing the concentration of an external reducing agent. The released plasmid DNA from the nanogels maintained intact structural integrity and exhibited appreciable gene transfection efficiency. The PEG/DNA nanogels could be potentially applied for gene therapy including DNA vaccination. PMID:17105212

Mok, Hyejung; Park, Tae Gwan

2006-01-01

184

Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing.  

PubMed

Standardization of DNA extraction is a fundamental issue of fidelity and comparability in investigations of environmental microbial communities. Commercial kits for soil or feces are often adopted for studies of activated sludge because of a lack of specific kits, but they have never been evaluated regarding their effectiveness and potential biases based on high throughput sequencing. In this study, seven common DNA extraction kits were evaluated, based on not only yield/purity but also sequencing results, using two activated sludge samples (two sub-samples each, i.e. ethanol-fixed and fresh, as-is). The results indicate that the bead-beating step is necessary for DNA extraction from activated sludge. The two kits without the bead-beating step yielded very low amounts of DNA, and the least abundant operational taxonomic units (OTUs), and significantly underestimated the Gram-positive Actinobacteria, Nitrospirae, Chloroflexi, and Alphaproteobacteria and overestimated Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, and the rare phyla whose cell walls might have been readily broken. Among the other five kits, FastDNA(@) SPIN Kit for Soil extracted the most and the purest DNA. Although the number of total OTUs obtained using this kit was not the highest, the abundant OTUs and abundance of Actinobacteria demonstrated its efficiency. The three MoBio kits and one ZR kit produced fair results, but had a relatively low DNA yield and/or less Actinobacteria-related sequences. Moreover, the 50 % ethanol fixation increased the DNA yield, but did not change the sequenced microbial community in a significant way. Based on the present study, the FastDNA SPIN kit for Soil is recommended for DNA extraction of activated sludge samples. More importantly, the selection of the DNA extraction kit must be done carefully if the samples contain dominant lysing-resistant groups, such as Actinobacteria and Nitrospirae. PMID:22760785

Guo, Feng; Zhang, Tong

2013-05-01

185

Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples  

PubMed Central

Background Technical advances following the Human Genome Project revealed that high-quality and -quantity DNA may be obtained from whole saliva samples. However, usability of previously collected samples and the effects of environmental conditions on the samples during collection have not been assessed in detail. In five studies we document the effects of sample volume, handling and storage conditions, type of collection device, and oral sampling location, on quantity, quality, and genetic assessment of DNA extracted from cells present in saliva. Methods Saliva samples were collected from ten adults in each study. Saliva volumes from .10-1.0 ml, different saliva collection devices, sampling locations in the mouth, room temperature storage, and multiple freeze-thaw cycles were tested. One representative single nucleotide polymorphism (SNP) in the catechol-0-methyltransferase gene (COMT rs4680) and one representative variable number of tandem repeats (VNTR) in the serotonin transporter gene (5-HTTLPR: serotonin transporter linked polymorphic region) were selected for genetic analyses. Results The smallest tested whole saliva volume of .10 ml yielded, on average, 1.43 ± .77 ?g DNA and gave accurate genotype calls in both genetic analyses. The usage of collection devices reduced the amount of DNA extracted from the saliva filtrates compared to the whole saliva sample, as 54-92% of the DNA was retained on the device. An "adhered cell" extraction enabled recovery of this DNA and provided good quality and quantity DNA. The DNA from both the saliva filtrates and the adhered cell recovery provided accurate genotype calls. The effects of storage at room temperature (up to 5 days), repeated freeze-thaw cycles (up to 6 cycles), and oral sampling location on DNA extraction and on genetic analysis from saliva were negligible. Conclusions Whole saliva samples with volumes of at least .10 ml were sufficient to extract good quality and quantity DNA. Using 10 ng of DNA per genotyping reaction, the obtained samples can be used for more than one hundred candidate gene assays. When saliva is collected with an absorbent device, most of the nucleic acid content remains in the device, therefore it is advisable to collect the device separately for later genetic analyses.

2011-01-01

186

High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR  

Microsoft Academic Search

We have developed an accurate, yet inexpensive and high-throughput, method for determining the allele frequency of biallelic polymorphisms in pools of DNA samples. The assay combines kinetic (real-time quantitative) PCR with allele-specific amplification and requires no post-PCR processing. The relative amounts of each allele in a sample are quantified. This is performed by dividing equal aliquots of the pooled DNA

Søren Germer; Michael J. Holland; Russell Higuchi

2000-01-01

187

Assessing the Value of DNA Barcodes for Molecular Phylogenetics: Effect of Increased Taxon Sampling in Lepidoptera  

PubMed Central

Background A common perception is that DNA barcode datamatrices have limited phylogenetic signal due to the small number of characters available per taxon. However, another school of thought suggests that the massively increased taxon sampling afforded through the use of DNA barcodes may considerably increase the phylogenetic signal present in a datamatrix. Here I test this hypothesis using a large dataset of macrolepidopteran DNA barcodes. Methodology/Principal Findings Taxon sampling was systematically increased in datamatrices containing macrolepidopteran DNA barcodes. Sixteen family groups were designated as concordance groups and two quantitative measures; the taxon consistency index and the taxon retention index, were used to assess any changes in phylogenetic signal as a result of the increase in taxon sampling. DNA barcodes alone, even with maximal taxon sampling (500 species per family), were not sufficient to reconstruct monophyly of families and increased taxon sampling generally increased the number of clades formed per family. However, the scores indicated a similar level of taxon retention (species from a family clustering together) in the cladograms as the number of species included in the datamatrix was increased, suggesting substantial phylogenetic signal below the ‘family’ branch. Conclusions/Significance The development of supermatrix, supertree or constrained tree approaches could enable the exploitation of the massive taxon sampling afforded through DNA barcodes for phylogenetics, connecting the twigs resolved by barcodes to the deep branches resolved through phylogenomics.

Wilson, John James

2011-01-01

188

Limitations and recommendations for successful DNA extraction from forensic soil samples: a review.  

PubMed

Soil is commonly used in forensic casework to provide discriminatory power to link a suspect to a crime scene. Standard analyses examine the intrinsic properties of soils, including mineralogy, geophysics, texture and colour; however, soils can also support a vast amount of organisms, which can be examined using DNA fingerprinting techniques. Many previous genetic analyses have relied on patterns of fragment length variation produced by amplification of unidentified taxa in the soil extract. In contrast, the development of advanced DNA sequencing technologies now provides the ability to generate a detailed picture of soil microbial communities and the taxa present, allowing for improved discrimination between samples. However, DNA must be efficiently extracted from the complex soil matrix to achieve accurate and reproducible DNA sequencing results, and extraction efficacy is highly dependent on the soil type and method used. As a result, a consideration of soil properties is important when estimating the likelihood of successful DNA extraction. This would include a basic understanding of soil components, their interactions with DNA molecules and the factors that affect such interactions. This review highlights some important considerations required prior to DNA extraction and discusses the use of common chemical reagents in soil DNA extraction protocols to achieve maximum efficacy. Together, the information presented here is designed to facilitate informed decisions about the most appropriate sampling and extraction methodology, relevant both to the soil type and the details of a specific forensic case, to ensure sufficient DNA yield and enable successful analysis. PMID:24796953

Young, Jennifer M; Rawlence, Nicolas J; Weyrich, Laura S; Cooper, Alan

2014-05-01

189

Simplified method for DNA and protein staining of human hematopoietic cell samples  

SciTech Connect

A rapid reproducible method yielding high resolution analysis of DNA and protein in human hematopoietic cell samples was developed by modification of the propidium iodide (PI) and fluorescein isothiocyanate (FITC) procedure. Cell staining involved sequential addition of each reagent (RNase, FITC, and PI) to ethanol-fixed cells and requires no centrifiguation steps. Stained cells are analyzed in the reagent solutions. Analysis of bone marrow samples from multiple myeloma patients revealed mixed 2C DNA and aneuploid populations with the aneuploid cells having a significantly higher protein content. This approach permitted differential cell cycle kinetic analysis of the 2C DNA and the aneuploid population.

Crissman, H.A.; Egmond, J.V.; Holdrinet, R.S.; Pennings, A.; Haanen, C.

1980-01-01

190

An Automated Sample Preparation System for Large-Scale DNA Sequencing  

PubMed Central

Recent advances in DNA sequencing technologies, both in the form of high lane-density gels and automated capillary systems, will lead to an increased requirement for sample preparation systems that operate at low cost and high throughput. As part of the development of a fully automated sequencing system, we have developed an automated subsystem capable of producing 10,000 sequence-ready ssDNA templates per day from libraries of M13 plaques at a cost of $0.29 per sample. This Front End has been in high throughput operation since June, 1997 and has produced > 400,000 high-quality DNA templates.

Marziali, Andre; Willis, Thomas D.; Federspiel, Nancy A.; Davis, Ronald W.

1999-01-01

191

Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution  

PubMed Central

Bisulfite sequencing measures absolute levels of DNA methylation at single-nucleotide resolution, providing a robust platform for molecular diagnostics. Here, we optimize bisulfite sequencing for genome-scale analysis of clinical samples. Specifically, we outline how restriction digestion targets bisulfite sequencing to hotspots of epigenetic regulation; we show that 30ng of DNA are sufficient for genome-scale analysis; we demonstrate that our protocol works well on formalin-fixed, paraffin-embedded (FFPE) samples; and we describe a statistical method for assessing significance of altered DNA methylation patterns.

Gu, Hongcang; Bock, Christoph; Mikkelsen, Tarjei S.; Jager, Natalie; Smith, Zachary D.; Tomazou, Eleni; Gnirke, Andreas; Lander, Eric S.; Meissner, Alexander

2010-01-01

192

Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution.  

PubMed

Bisulfite sequencing measures absolute levels of DNA methylation at single-nucleotide resolution, providing a robust platform for molecular diagnostics. We optimized bisulfite sequencing for genome-scale analysis of clinical samples: here we outline how restriction digestion targets bisulfite sequencing to hotspots of epigenetic regulation and describe a statistical method for assessing significance of altered DNA methylation patterns. Thirty nanograms of DNA was sufficient for genome-scale analysis and our protocol worked well on formalin-fixed, paraffin-embedded samples. PMID:20062050

Gu, Hongcang; Bock, Christoph; Mikkelsen, Tarjei S; Jäger, Natalie; Smith, Zachary D; Tomazou, Eleni; Gnirke, Andreas; Lander, Eric S; Meissner, Alexander

2010-02-01

193

Estimating Animal Abundance Using Noninvasive DNA Sampling: Promise and Pitfalls  

Microsoft Academic Search

Advances in molecular biology offer promise to the study of demographic characteristics of rare or hard-to-capture species, because individuals can now be identified through noninvasive sampling such as fecal collection or hair snags. However, individual genotyping using such methods currently leads to a novel problem that we call a ''shadow effect,'' because some animals not captured previously are believed to

L. Scott Mills; John J. Citta; Kevin P. Lair; Michael K. Schwartz; David A. Tallmon

2000-01-01

194

A Nuclear Protein Involved in Apoptotic-like DNA Degradation in Stylonychia: Implications for Similar Mechanisms in Differentiating and Starved Cells  

PubMed Central

Ciliates are unicellular eukaryotic organisms containing two types of nuclei: macronuclei and micronuclei. After the sexual pathway takes place, a new macronucleus is formed from a zygote nucleus, whereas the old macronucleus is degraded and resorbed. In the course of macronuclear differentiation, polytene chromosomes are synthesized that become degraded again after some hours. Most of the DNA is eliminated, and the remaining DNA is fragmented into small DNA molecules that are amplified to a high copy number in the new macronucleus. The protein Pdd1p (programmed DNA degradation protein 1) from Tetrahymena has been shown to be present in macronuclear anlagen in the DNA degradation stage and also in the old macronuclei, which are resorbed during the formation of the new macronucleus. In this study the identification and localization of a Pdd1p homologous protein in Stylonychia (Spdd1p) is described. Spdd1p is localized in the precursor nuclei in the DNA elimination stage and in the old macronuclei during their degradation, but also in macronuclei and micronuclei of starved cells. In all of these nuclei, apoptotic-like DNA breakdown was detected. These data suggest that Spdd1p is a general factor involved in programmed DNA degradation in Stylonychia.

Maercker, Christian; Kortwig, Heike; Nikiforov, Mikhail A.; Allis, C. David; Lipps, Hans J.

1999-01-01

195

DNA extraction and amplification of 10-day, room-temperature blood samples.  

PubMed

DNA was serially studied in 20 samples of buffy coat stored at room temperature. Each sample was divided into 5 equal volumes, namely D0, D3, D5, D7 and D10. DNA extraction was performed on days 0, 3, 5, 7 and 10 after blood collection. The mean ratio of OD260/OD280 of the DNA obtained from D0 to D10 ranged from 1.77 to 1.79, and the mean amounts of the DNA obtained from D0 to D10 ranged from 602 to 740 ng/ul. There were no significant differences in the mean ratio and amounts of DNA obtained among these samples (p > 0.05). Subsequently, amplification was successfully performed from this template DNA to yield products of 1.4 kb and 142 bp at the sites associated with beta globin and factor VIII genes, respectively. These findings suggest the possibility of sending blood samples for DNA analysis by mail, or no ice is required during transportation. PMID:10730541

Sasanakul, W; Chuansumrit, A; Rurgkhum, S; Udomsubpayakul, U; Hathirat, P

1999-11-01

196

Preprocessing and Quality Control Strategies for Illumina DASL Assay-Based Brain Gene Expression Studies with Semi-Degraded Samples.  

PubMed

Available statistical preprocessing or quality control analysis tools for gene expression microarray datasets are known to greatly affect downstream data analysis, especially when degraded samples, unique tissue samples, or novel expression assays are used. It is therefore important to assess the validity and impact of the assumptions built in to preprocessing schemes for a dataset. We developed and assessed a data preprocessing strategy for use with the Illumina DASL-based gene expression assay with partially degraded postmortem prefrontal cortex samples. The samples were obtained from individuals with autism as part of an investigation of the pathogenic factors contributing to autism. Using statistical analysis methods and metrics such as those associated with multivariate distance matrix regression and mean inter-array correlation, we developed a DASL-based assay gene expression preprocessing pipeline to accommodate and detect problems with microarray-based gene expression values obtained with degraded brain samples. Key steps in the pipeline included outlier exclusion, data transformation and normalization, and batch effect and covariate corrections. Our goal was to produce a clean dataset for subsequent downstream differential expression analysis. We ultimately settled on available transformation and normalization algorithms in the R/Bioconductor package lumi based on an assessment of their use in various combinations. A log2-transformed, quantile-normalized, and batch and seizure-corrected procedure was likely the most appropriate for our data. We empirically tested different components of our proposed preprocessing strategy and believe that our results suggest that a preprocessing strategy that effectively identifies outliers, normalizes the data, and corrects for batch effects can be applied to all studies, even those pursued with degraded samples. PMID:22375143

Chow, Maggie L; Winn, Mary E; Li, Hai-Ri; April, Craig; Wynshaw-Boris, Anthony; Fan, Jian-Bing; Fu, Xiang-Dong; Courchesne, Eric; Schork, Nicholas J

2012-01-01

197

Confirmation of cadaveric blood sample identity by DNA profiling using Short Tandem Repeat (STR) analysis.  

PubMed

Blood samples collected from deceased tissue donors for mandatory transfusion microbiology testing may be taken either at the time of tissue donation, or residual samples may be retrieved from hospital laboratories where they were originally used for ante-mortem tests. In the latter case, sample labelling may not conform to the required standard, which stipulates that three independent identifiers be provided. If no alternative adequately labelled sample is available for testing the donated tissues may have to be discarded, which can adversely affect tissue sufficiency. An alternative method to ensure that the blood sample to be tested is from the intended deceased donor is to confirm the identity of the blood sample by Deoxyribonucleic Nucleic Acid (DNA) Short Tandem Repeats (STR) analysis, then comparing the DNA profile with the DNA from the donated tissues. If the two DNA profiles are identical, probability calculations can demonstrate the chance of the two samples of DNA being from the same or different individuals. The authors have used this approach to salvage deceased tissue donations. PMID:18483780

Warwick, Ruth M; Rushambuza, F G; Brown, J; Patel, R; Tabb, S; Poniatowski, S; Ranson, A J; Brown, C J

2008-12-01

198

Diagnosis and quantitative detection of HSV DNA in samples from patients with suspected herpes simplex encephalitis.  

PubMed

Diagnosis of herpes simplex encephalitis (HSE) is based on the detection of herpes simplex virus (HSV) DNA in patients' CSF samples. HSV DNA quantitation has the potential for estimating the effects of antiviral therapy. The aim of this study was to diagnose HSV DNA in HSE suspected patients and the quantitative analysis of its genome using real-time PCR to assess the value of the viral load in the course of antiviral treatment. The CSF samples were collected from 236 consecutive HSE suspected patients from November 2004 to May 2008. Upon DNA extraction, the samples were analyzed by Real-Time PCR assay. A set of primers amplified a common sequence of HSV glycoprotein B gene. The copy numbers of unknown samples were expressed via a standard curve drawn with a known amount of amplified cloned plasmid. Of the 236 samples, 137 (58%) came from males and 99 (42%) from females. The HSV genome was detected in 22 (9.3%) patients by PCR, 13 males/ 9 females. Serial CSF samples were available from 10 of the 22 patients. The range of the HSV DNA copy numbers in the clinical samples ranged from 2.5 × 10² to 1.7 × 10? copies/mL of CSF. Quantitative PCR results can be helpful in evaluating the efficacy of antiviral therapy in the above-mentioned patients. There is an association between the initial viral load and the duration of treatment course. PMID:21670919

Ziyaeyan, Mazyar; Alborzi, Abdolvahab; Borhani Haghighi, Afshin; Jamalidoust, Marziyeh; Moeini, Mahsa; Pourabbas, Bahman

2011-01-01

199

Current sample handling methods for measurement of platinum-DNA adducts in leucocytes in man lead to discrepant results in DNA adduct levels and DNA repair  

Microsoft Academic Search

DNA adduct levels were measured with atomic spectroscopy in white blood cells (WBCs) from patients with solid tumours who were treated with six weekly courses of cisplatin. In 21 patients (I) the WBCs were collected after thawing frozen whole-blood samples according to a previously described method. In 32 other patients (II) WBCs were collected immediately after blood sample collection. The

J Ma; J Verweij; ASTh Planting; M de Boer-Dennert; HE van Ingen; MEL van der Burg; G Stoter; JHM Schellens

1995-01-01

200

Detection of Echinococcus multilocularis in faeces by nested PCR with the use of diluted DNA samples.  

PubMed

The aim of this study was to choose the optimal variant of PCR examination of faeces to detect Echinococcus multilocularis infection which would allow to reduce the influence of different inhibitors in faeces. The investigation was carried out by comparison of 3 different methods of DNA isolation from faeces and different DNA dilutions used in PCR. Thirty five intestines of red foxes were used. Small intestines were examined by the sedimentation and counting technique (SCT). Faeces were collected from the rectum for PCR and flotation. DNA were isolated with the use of 3 different methods. Two methods were dedicated for faeces: method 1 (M1)--for larger samples and method 2 (M2) - for standard samples. The third method, method 3 (M3), was not dedicated for faeces. DNA samples were tested by nested PCR in 6 variants: not diluted (1/1) and 5 diluted (1/2.5, 1/5, 1/10. 1/20, 1/40). E. multilocularis was found by SCT in 18 from 35 (51.4%) intestines. Taenia-type eggs were detected only in 20.0% of faecal samples. In PCR the highest number of positive results (45.7%) were obtained during examination of DNA isolated by M1 method, and then 40.0% and 34.3%, respectively, for M2 and M3. In some samples positive results in PCR were obtained only in diluted DNA. For example, 8 from 12 positive samples isolated by M3 method gave the PCR negative results in non-diluted DNA and positive only after dilution 1:2.5, 1:10 or 1:20. Also 3 samples isolated by methods dedicated for stool gave positive results only after DNA dilution. The investigation has revealed that in copro-PCR for detection of E. multilocularis infection additional using of diluted DNA (besides non diluted) can avoid false negative results causing by PCR inhibition. In the best method of DNA isolation (M1), the use of non diluted DNA sample together with diluted in proportion 1:10 seems to be optimal. PMID:24724473

Karamon, J

2014-01-01

201

Hematoporphyrin-sensitized degradation of deoxyribose and DNA in high intensity near-UV picosecond pulsed laser photolysis  

NASA Astrophysics Data System (ADS)

The photosensitized degradation of deoxyribose and DNA, using hematoporphyrin (HP) and picosecond laser pulses at high intensities (pulse duration 30 ps, ? exc = 355 nm, light intensity range 10 8-10 10W/cm 2) was studied. Aldehyde formation from 2-deoxy- D-ribose and long-chain double-stranded DNA, when analyzed as a function of light intensity, followed a non-linear dependence, suggesting the involvement of multiphoton light absorption by HP. The degradation mechanism was studied by analysis of the yield dependence on excitation intensity and the effect of added radical scavengers. The participation of OH radicals in the degradation process was confirmed by spin trapping techniques. At low light intensities added N 2O largely increased product formation, suggesting that HP photoionization predominates under these conditions. At higher intensities (I ? 3 GW/cm 2) the product yield was not affected by N 2O which, combined with spin trapping data, suggested that OH radical formation occurred, but that neither HP photoionization nor peroxy radical formation was involved. Single and double strand breaks in supercoiled plasmid DNA (pBR 322) confirmed the generation of OH or OH-like radicals during high-intensity excitation of HP. A mechanism involving a multistep excitation of HP, followed by resonance energy transfer to H 2O resulting in dissociation to yield OH and H atoms, is proposed.

Gantchev, Tsvetan G.; Grabner, Gotfried; Keskinova, Elka; Angelov, Dimitr; van Lier, Johan E.

1995-01-01

202

Species-Specific Identification from Incomplete Sampling: Applying DNA Barcodes to Monitoring Invasive Solanum Plants  

PubMed Central

Comprehensive sampling is crucial to DNA barcoding, but it is rarely performed because materials are usually unavailable. In practice, only a few rather than all species of a genus are required to be identified. Thus identification of a given species using a limited sample is of great importance in current application of DNA barcodes. Here, we selected 70 individuals representing 48 species from each major lineage of Solanum, one of the most species-rich genera of seed plants, to explore whether DNA barcodes can provide reliable specific-species discrimination in the context of incomplete sampling. Chloroplast genes ndhF and trnS-trnG and the nuclear gene waxy, the commonly used markers in Solanum phylogeny, were selected as the supplementary barcodes. The tree-building and modified barcode gap methods were employed to assess species resolution. The results showed that four Solanum species of quarantine concern could be successfully identified through the two-step barcoding sampling strategy. In addition, discrepancies between nuclear and cpDNA barcodes in some samples demonstrated the ability to discriminate hybrid species, and highlights the necessity of using barcode regions with different modes of inheritance. We conclude that efficient phylogenetic markers are good candidates as the supplementary barcodes in a given taxonomic group. Critically, we hypothesized that a specific-species could be identified from a phylogenetic framework using incomplete sampling–through this, DNA barcoding will greatly benefit the current fields of its application.

Zhang, Wei; Fan, Xiaohong; Zhu, Shuifang; Zhao, Hong; Fu, Lianzhong

2013-01-01

203

An efficient protocol for tissue sampling and DNA isolation from the stem bark of Leguminosae trees.  

PubMed

Traditionally, molecular studies of plant species have used leaves as the source of DNA. However, sampling leaves from tall tree species can be quite difficult and expensive. We developed a sequence of procedures for using stem bark as a source of DNA from Leguminosae trees of the Atlantic Forest and the Cerrado. Leguminosae is an important species-rich family in these two highly diverse and endangered biomes. A modified CTAB protocol for DNA isolation is described, and details of the procedures for sampling and storage of the bark are given. The procedures were initially developed for three species, and then their applicability for 15 other species was evaluated. DNA of satisfactory quality was obtained from the bark of all species. The amounts of DNA obtained from leaves were slightly higher than from bark samples, while its purity was the same. Storing the bark frozen or by drying in silica gel yielded similar results. Polymerase chain reaction amplification worked for both plastid and nuclear genomes. This alternative for isolating DNA from bark samples of trees facilitates field work with these tree species. PMID:19283676

Novaes, R M L; Rodrigues, J G; Lovato, M B

2009-01-01

204

Human sperm DNA integrity in normal and abnormal semen samples and its correlation with sperm characteristics.  

PubMed

Reports indicate an increase in the incidence of DNA fragmentation in male factor infertility and its role in the outcome of assisted reproductive techniques (ART). However, reports are conflicting between the relationships of sperm DNA integrity with conventional semen parameters. We examined the relationship between conventional sperm parameters and DNA integrity using acridine orange (AO) test. The study included 373 patients and 28 fertile volunteers. DNA normality was compared with semen parameters between the patient and donor populations. Significant correlations were noted between DNA normality and sperm concentration (r = 0.18, P = 0.000), motility (r = 0.21, P = 0.0001), rapid motility (0.19, P = 0.000), normal morphology by World Health Organization (r = 0.15, P = 0.019) and head defects (r = -0.15, P = 0.023). A significant difference was noted in AO levels between donors and patients with asthenozoospermia (P = 0.002) and oligoasthenozoospermia (P = 0.001). A significant difference in DNA integrity was noted in samples having <30% and >30% normal morphology. A wide range of % DNA normality was observed in the patient group. Sperm assessment for DNA status using AO is reliable and shows good correlation with sperm count, motility and morphology. Assessment of sperm DNA status with AO staining may be helpful prior to ART. PMID:19601931

Varghese, A C; Bragais, F M; Mukhopadhyay, D; Kundu, S; Pal, M; Bhattacharyya, A K; Agarwal, A

2009-08-01

205

Chemical Characterization of Polynuclear Aromatic Hydrocarbon Degradation Products from Sampling Artifacts.  

National Technical Information Service (NTIS)

The objective of the study was to characterize the polar components, mainly polynuclear aromatic hydrocarbon (PAH) derivatives, in air samples and to determine whether these compounds are from sampling artifacts or from the sampled air. A literature surve...

J. C. Chuang S. W. Hannan L. E. Slivon

1987-01-01

206

Detecting and quantifying lewisite degradation products in environmental samples using arsenic speciation  

SciTech Connect

This report describes a unique method for identifying and quantifying lewisite degradation products using arsenic (III) and arsenic (IV) speciation in solids and in solutions. Gas chromatographic methods, as well as high-performance liquid chromatographic methods are described for separation of arsenic species. Inductively coupled plasma-mass spectrographic methods are presented for the detection of arsenic.

Bass, D.A.; Yaeger, J.S.; Kiely, J.T.; Crain, J.S.; Shem, L.M.; O`Neill, H.J.; Gowdy, M.J. [Argonne National Lab., IL (United States); Besmer, M.; Mohrman, G.B. [Rocky Mountain Arsenal, Commerce City, CO (United States)

1995-12-31

207

Double-degradable responsive self-assembled multivalent arrays--temporary nanoscale recognition between dendrons and DNA.  

PubMed

This article reports self-assembling dendrons which bind DNA in a multivalent manner. The molecular design directly impacts on self-assembly which subsequently controls the way these multivalent nanostructures bind DNA--this can be simulated by multiscale modelling. Incorporation of an S-S linkage between the multivalent hydrophilic dendron and the hydrophobic units responsible for self-assembly allows these structures to undergo triggered reductive cleavage, with dithiothreitol (DTT) inducing controlled breakdown, enabling the release of bound DNA. As such, the high-affinity self-assembled multivalent binding is temporary. Furthermore, because the multivalent dendrons are constructed from esters, a second slow degradation step causes further breakdown of these structures. This two-step double-degradation mechanism converts a large self-assembling unit with high affinity for DNA into small units with no measurable binding affinity--demonstrating the advantage of self-assembled multivalency (SAMul) in achieving highly responsive nanoscale binding of biological targets. PMID:24263553

Barnard, Anna; Posocco, Paola; Fermeglia, Maurizio; Tschiche, Ariane; Calderon, Marcelo; Pricl, Sabrina; Smith, David K

2014-01-21

208

Hypoxic activation of ATR and the suppression of the initiation of DNA replication through cdc6 degradation.  

PubMed

Many severely hypoxic cells fail to initiate DNA replication, but the mechanism underlying this observation is unknown. Specifically, although the ataxia-telangiectasia-rad3 related (ATR) kinase has been shown to be activated in hypoxic cells, several studies have not been able to document down-stream consequences of ATR activation in these cells. By clearly defining the DNA replication initiation checkpoint in hypoxic cells, we now demonstrate that ATR is responsible for activating this checkpoint. We show that the hypoxic activation of ATR leads to the phosphorylation-dependent degradation of the cdc25a phosphatase. Downregulation of cdc25a protein by ATR in hypoxic cells decreases CDK2 phosphorylation and activity, which results in the degradation of cdc6 by APC/C(Cdh1). These events do not occur in hypoxic cells when ATR is depleted, and the initiation of DNA replication is maintained. We therefore present a novel mechanism of cdc6 regulation in which ATR can have a central role in inhibiting the initiation of DNA replication by the regulation of cdc6 by APC/C(Cdh1). This model provides insight into the biology and therapy of hypoxic tumors. PMID:22179839

Martin, L; Rainey, M; Santocanale, C; Gardner, L B

2012-09-01

209

Sources of Pre-Analytical Variations in Yield of DNA Extracted from Blood Samples: Analysis of 50,000 DNA Samples in EPIC  

Microsoft Academic Search

The European Prospective Investigation into Cancer and nutrition (EPIC) is a long-term, multi-centric prospective study in Europe investigating the relationships between cancer and nutrition. This study has served as a basis for a number of Genome-Wide Association Studies (GWAS) and other types of genetic analyses. Over a period of 5 years, 52,256 EPIC DNA samples have been extracted using an

Elodie Caboux; Christophe Lallemand; Gilles Ferro; Bertrand Hémon; Maimuna Mendy; Carine Biessy; Matt Sims; Nick Wareham; Abigail Britten; Anne Boland; Amy Hutchinson; Afshan Siddiq; Paolo Vineis; Elio Riboli; Isabelle Romieu; Sabina Rinaldi; Marc J. Gunter; Petra H. M. Peeters; Yvonne T. van der Schouw; Ruth Travis; H. Bas Bueno-de-Mesquita; Federico Canzian; Maria-José Sánchez; Guri Skeie; Karina Standahl Olsen; Eiliv Lund; Roberto Bilbao; Núria Sala; Aurelio Barricarte; Domenico Palli; Carmen Navarro; Salvatore Panico; Maria Luisa Redondo; Silvia Polidoro; Laure Dossus; Marie Christine Boutron-Ruault; Françoise Clavel-Chapelon; Antonia Trichopoulou; Dimitrios Trichopoulos; Pagona Lagiou; Heiner Boeing; Eva Fisher; Rosario Tumino; Claudia Agnoli; Pierre Hainaut

2012-01-01

210

A comparison of six methods for genomic DNA extraction suitable for PCR-based genotyping applications using ovine milk samples  

Microsoft Academic Search

Isolation of amplifiable genomic DNA is a prerequisite for the genetic assessment of diseases and disease susceptibility in farm animals. Milk somatic cells are a practical, animal friendly and cost-effective source of genomic DNA in milking ruminants. In this study, six different DNA extraction methods were optimized, evaluated and compared for the isolation of DNA from ovine milk samples. Methods

Androniki Psifidi; Chrysostomos I. Dovas; Georgios Banos

2010-01-01

211

Effects of cesium ions and cesium vapor on selected ATS-F samples. [thermal control coating degradation  

NASA Technical Reports Server (NTRS)

Thermal control coating samples were subjected to cesium ion beam and vapor exposures. Degradation of solar absorptance and infrared emittance were measured. Solar cells and samples selected from surfaces on the ATS-F spacecraft likely to experience ion or vapor impingement were bombarded by 10-volt cesium ions. Other samples were subjected to high levels of cesium vapor. Aluminum and white paint were backsputtered by 550-volt cesium ions onto selected samples. For direct bombardment, the threshold for ion-induced property changes was above five-thousand trillion ions/sq cm. With material sputtered from a 450-sq cm target onto samples 36 cm distant, the threshold for noticeable effects was above 5 times 10 to the 17-th power ions/sq cm.

Kemp, R. F.; Beynon, J. C.; Hall, D. F.; Luedke, E. E.

1973-01-01

212

Nuclear translocation of p21{sup WAF1/CIP1} protein prior to its cytosolic degradation by UV enhances DNA repair and survival  

SciTech Connect

We previously reported that UV induced rapid proteasomal degradation of p21 protein in an ubiquitination-independent manner. Here, UV-induced p21 proteolysis was found to occur in the cytosol. Before cytosolic degradation, however, p21 protein translocated to and transiently accumulated in the nucleus. Nuclear translocation of p21 was not required for its degradation, but rather promoted DNA repair and cell survival. Overexpression of the wild type p21, but not the one with defective nuclear localization signal (NLS), reduced UV-induced DNA damage and cell death. Some of p21 protein translocated to the nucleus were associated with chromatin-bound PCNA and saved from UV-induced proteolysis. These data together show that p21 translocates to the nucleus to participate in DNA repair, while the rest is rapidly degraded in the cytosol. We propose that our findings reflect a mechanism to facilitate removal of damaged cells, enhancing DNA repair at the same time.

Lee, Ji Young; Kim, Hee Suk; Kim, Joo Young [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of)] [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Sohn, Jeongwon, E-mail: biojs@korea.ac.kr [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of)] [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of)

2009-12-25

213

DNA-Methylation Profiling of Fetal Tissues Reveals Marked Epigenetic Differences between Chorionic and Amniotic Samples  

PubMed Central

Epigenetic mechanisms including DNA methylation are supposed to play a key role in fetal development. Here we have investigated fetal DNA-methylation levels of 27,578 CpG loci in 47 chorionic villi (CVS) and 16 amniotic cell (AC) samples. Methylation levels differed significantly between karyotypically normal AC and CVS for 2,014 genes. AC showed more extreme DNA-methylation levels of these genes than CVS and the differentially methylated genes are significantly enriched for processes characteristic for the different cell types sampled. Furthermore, we identified 404 genes differentially methylated in CVS with trisomy 21. These genes were significantly enriched for high CG dinucleotid (CpG) content and developmental processes associated with Down syndrome. Our study points to major tissue-specific differences of fetal DNA-methylation and gives rise to the hypothesis that part of the Down syndrome phenotype is epigenetically programmed in the first trimester of pregnancy.

Eckmann-Scholz, Christel; Bens, Susanne; Kolarova, Julia; Schneppenheim, Sina; Caliebe, Almuth; Heidemann, Simone; von Kaisenberg, Constantin; Kautza, Monika; Jonat, Walter; Siebert, Reiner; Ammerpohl, Ole

2012-01-01

214

Linear Mixture Analysis: A Mathematical Approach to Resolving Mixed DNA Samples  

Microsoft Academic Search

Abstract With the advent of PCR-based STR typing systems, mixed samples can be separated into their individual DNA profiles. Quantitative peak,information can help in this analysis. However, despite such advances, forensic mixture analysis still remains a laborious art, with the high cost and effort often precluding timely reporting. We introduce here a new automated,approach,to resolving forensic DNA mixtures. Our

Mark W. Perlin; Beata Szabady

2001-01-01

215

An evaluation of long-term preservation methods for brown bear ( Ursus arctos ) faecal DNA samples  

Microsoft Academic Search

Relatively few large-scale faecal DNA studieshave been initiated due to difficulties inamplifying low quality and quantity DNAtemplate. To improve brown bear faecal DNA PCRamplification success rates and to determinepost collection sample longevity, fivepreservation methods were evaluated: 90%ethanol, DETs buffer, silica-dried, oven-driedstored at room temperature, and oven-driedstored at -20 °C. Preservationeffectiveness was evaluated for 50 faecalsamples by PCR amplification of a

Melanie A. Murphy; Lisette P. Waits; Katherine C. Kendall; Samuel K. Wasser; Jerry A. Higbee; Robert Bogden

2002-01-01

216

Detection of Legionella DNA in human and guinea pig urine samples by the polymerase chain reaction  

Microsoft Academic Search

A detection system forLegionella DNA in urine samples based on the polymerase chain reaction (PCR) was developed and tested on infected guinea pigs and patients suffering from pneumonia. Results were compared with standard methods for diagnosis of Legionnaires' disease. A primer system was selected which amplifies a 108 bp DNA fragment of the 5S rRNA gene. The sensitivity of the

M. Maiwaldl; M. Schill; C. Stockinger; J. H. Helbig; P. C. Lück; W. Witzleb; H.-G. Sonntag

1995-01-01

217

Self-Contained, Fully Integrated Biochips for Sample Preparation, PCR Amplification and DNA Microarray Analysis  

Microsoft Academic Search

Rapid developments in back-end detection platforms (such as DNA microarrays, capillary electrophoresis, real-time polymerase\\u000a chain reaction and mass spectroscopy) for genetic analysis have shifted the bottleneck to front-end sample preparation where\\u000a the ‘real’ samples are used. In this chapter, we present a fully integrated biochip device that can perform on-chip sample\\u000a preparation (including magnetic bead-based cell capture, cell preconcentration and

Robin Hui Liu; Piotr Grodzinski; Jianing Yang; Ralf Lenigk

218

Mitochondrial DNA control region variation from samples of the Moroccan population.  

PubMed

In an effort to facilitate forensic mitochondrial DNA (mtDNA) testing in Morocco, high-quality control region sequences from 509 individuals were generated using a comprehensive processing and data review system. This large dataset of random samples from various Moroccan population groups (Arab speaking, Berber speaking, and Sahrawi speaking) exhibited a low random match probability (0.52 %) and a mean of pairwise comparisons of 13.24. The Moroccan mtDNA gene pool studied here was defined entirely by West Eurasian (58.15 %) and African haplogroups (41.85 %). PMID:23283404

Aboukhalid, Rachid; Sturk-Andreaggi, Kimberly; Bouabdellah, Mehdi; Squalli, Driss; Irwin, Jodi A; Amzazi, Saaïd

2013-07-01

219

Detection of the DNA of Borrelia afzelii, Anaplasma phagocytophilum and Babesia canis in blood samples from dogs in Warsaw  

Microsoft Academic Search

Each month, from March 2003 to February 2004, 34 blood samples from dogs were randomly selected from the blood samples delivered to two veterinary laboratories in Warsaw and tested for the DNA of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Babesia canis and Hepatozoon canis. Borrelia DNA was detected in seven of the 408 dogs, A phagocytophilum DNA was found in

W. Zygner; P. Górski; H. W?drychowicz

2009-01-01

220

Lack of DNA degradation in target cells lysed by granules derived from cytolytic T lymphocytes.  

PubMed

It has been shown previously that fragmentation of target cell DNA is an early event in lysis mediated by cytolytic T lymphocytes (CTL). In this study, we have investigated whether CTL-derived granules that exhibit lytic activity also induce DNA fragmentation in murine target cells. Cytolytic granules isolated from three different alloreactive CTL clones were tested for the induction of DNA fragmentation in P815 and EL4 target cells, by using a Triton X-100-facilitated, radiolabeled DNA release assay. In contrast to the CTL clones from which they were derived, the cytolytic granules did not induce DNA fragmentation. Agarose gel electrophoretic analysis of DNA confirmed the lack of discrete DNA fragments in target cells lysed by CTL-derived granules. Possible explanations for the difference in the ability of CTL and CTL-derived granules to trigger DNA fragmentation are discussed. PMID:3260910

Gromkowski, S H; Brown, T C; Masson, D; Tschopp, J

1988-08-01

221

Novel path to apoptosis: small transmembrane pores created by staphylococcal alpha-toxin in T lymphocytes evoke internucleosomal DNA degradation.  

PubMed Central

Peripheral-blood human T lymphocytes were treated with Staphylococcus aureus alpha-toxin. Membrane permeabilization was assessed by measuring efflux of K+ and Rb+ and influx of Na+, Ca2+, and propidium iodide. Cellular ATP and [3H]thymidine incorporation following lectin stimulation were measured as parameters for cell viability. Internucleosomal cleavage characteristic of programmed cell death was assessed by agarose gel electrophoresis and by quantifying low-molecular-weight, [3H]thymidine-labeled DNA fragments. Nanomolar concentrations of alpha-toxin evoked protracted, irreversible ATP depletion in both activated and resting T lymphocytes. Toxin-damaged cells also lost their ability to incorporate [3H]thymidine upon subsequent stimulation with phytohemagglutinin. These cells carried toxin hexamers, and their plasma membranes became permeable for monovalent ions but not for Ca2+ and propidium iodide. The permeabilization event was followed by internucleosomal DNA degradation characteristic of programmed cell death. Membranes of cells treated with high toxin doses (> 300 nM) became permeable to both Ca2+ and propidium iodide. In this case, ATP depletion occurred within minutes and no DNA degradation was observed. When cells were suspended in Na(+)-free buffer, alpha-toxin applied at low doses still bound and formed hexamers. However, these cells displayed neither DNA degradation nor loss of viability. The data indicate that formation of very small but not of large alpha-toxin pores may trigger programmed cell death in lymphocytes and that uncontrolled flux of Na+ ions may be an important event precipitating the suicide cascade. Images

Jonas, D; Walev, I; Berger, T; Liebetrau, M; Palmer, M; Bhakdi, S

1994-01-01

222

Orchestration of cooperative events in DNA synthesis and repair mechanism unraveled by transition path sampling of DNA polymerase 's closing  

NASA Astrophysics Data System (ADS)

Our application of transition path sampling to a complex biomolecular system in explicit solvent, the closing transition of DNA polymerase , unravels atomic and energetic details of the conformational change that precedes the chemical reaction of nucleotide incorporation. The computed reaction profile offers detailed mechanistic insights into, as well as kinetic information on, the complex process essential for DNA synthesis and repair. The five identified transition states extend available experimental and modeling data by revealing highly cooperative dynamics and critical roles of key residues (Arg-258, Phe-272, Asp-192, and Tyr-271) in the enzyme's function. The collective cascade of these sequential conformational changes brings the DNA/DNA polymerase system to a state nearly competent for the chemical reaction and suggests how subtle residue motions and conformational rate-limiting steps affect reaction efficiency and fidelity; this complex system of checks and balances directs the system to the chemical reaction and likely helps the enzyme discriminate the correct from the incorrect incoming nucleotide. Together with the chemical reaction, these conformational features may be central to the dual nature of polymerases, requiring specificity (for correct nucleotide selection) as well as versatility (to accommodate different templates at every step) to maintain overall fidelity. Besides leading to these biological findings, our developed protocols open the door to other applications of transition path sampling to long-time, large-scale biomolecular reactions.

Radhakrishnan, Ravi; Schlick, Tamar

2004-04-01

223

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

PubMed Central

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.

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

2012-01-01

224

REV7 is required for anaphase-promoting complex-dependent ubiquitination and degradation of translesion DNA polymerase REV1.  

PubMed

REV1 is a Y-family polymerase specialized for replicating across DNA lesions at the stalled replication folk. Due to the high error rate of REV1-dependent translesion DNA synthesis (TLS), tight regulation of REV1 activity is essential. Here, we show that human REV1 undergoes proteosomal degradation mediated by the E3 ubiquitin ligase known as anaphase-promoting complex (APC). REV1 associates with APC. Overexpression of APC coactivator CDH1 or CDC20 promotes polyubiquitination and proteosomal degradation of REV1. Surprisingly, polyubiquitination of REV1 also requires REV7, a TLS accessory protein that interacts with REV1 and other TLS polymerases. The N-terminal region of REV1 contains both the APC degron and an additional REV7-binding domain. Depletion of REV7 by RNA interference stabilizes REV1 by preventing polyubiquitination, whereas overexpression of REV7 augments REV1 degradation. Taken together, our findings suggest a role of REV7 in governing REV1 stability and interplay between TLS and APC-dependent proteolysis. PMID:23287467

Chun, Abel Chiu-Shun; Kok, Kin-Hang; Jin, Dong-Yan

2013-01-15

225

Design and biophysical characterization of bioresponsive degradable poly(dimethylaminoethyl methacrylate) based polymers for in vitro DNA transfection.  

PubMed

Water-soluble, degradable polymers based on poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) with low cytotoxicity and good p-DNA transfection efficiency are highlighted in this article. To solve the nondegradability issue of PDMAEMA, new polymers based on DMAEMA and 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) for gene transfection were synthesized. A poly(ethylene oxide) (PEO) azo-initiator was used as free-radical initiator. PEGylation was performed to improve water solubility and to reduce cytotoxicity of the polymers. The resulting polymers contain hydrolyzable ester linkages in the backbone and were soluble in water even with very high amounts of ester linkages. These degradable copolymers showed significantly less toxicity with a MTT assay using L929 cell lines and demonstrated promising DNA transfection efficiency when compared with the gold standard poly(ethyleneimine). Bioresponsive properties of the corresponding quaternized DMAEMA based degradable polymers were also studied. Although the quaternized DMAEMA copolymers showed enhanced water solubility, they were inferior in gene transfection and toxicity as compared to the unquaternized copolymers. PMID:22191470

Zhang, Yi; Zheng, Mengyao; Kissel, Thomas; Agarwal, Seema

2012-02-13

226

Powerful bacterial killing by buckwheat honeys is concentration-dependent, involves complete DNA degradation and requires hydrogen peroxide  

PubMed Central

Exposure of bacterial cells to honey inhibits their growth and may cause cell death. Our previous studies showed a cause-effect relationship between hydroxyl radical generated from honey hydrogen peroxide and growth arrest. Here we explored the role of hydroxyl radicals as inducers of bacterial cells death. The bactericidal effect of ·OH on antibiotic-resistant clinical isolates of MRSA and VRE and standard bacterial strains of E. coli and B. subtiles was examined using a broth microdilution assay supplemented with 3?-(p-aminophenyl) fluorescein (APF) as the ·OH trap, followed by colony enumeration. Bactericidal activities of eight honeys (six varieties of buckwheat, blueberry and manuka honeys) were analyzed. The MBC/MIC ratio ?4 and the killing curves indicated that honeys exhibited powerful, concentration-dependent bactericidal effect. The extent of killing depended on the ratio of honey concentration to bacterial load, indicating that honey dose was critical for its bactericidal efficacy. The killing rate and potency varied between honeys and ranged from over a 6-log10 to 4-log10 CFU/ml reduction of viable cells, equivalent to complete bacterial eradication. The maximal killing was associated with the extensive degradation of bacterial DNA. Honey concentration at which DNA degradation occurred correlated with cell death observed in the concentration-dependent cell-kill on agar plates. There was no quantitative relationship between the ·OH generation by honey and bactericidal effect. At the MBC, where there was no surviving cells and no DNA was visible on agarose gels, the ·OH levels were on average 2–3x lower than at Minimum Inhibitory Concentration (MICs) (p < 0.0001). Pre-treatment of honey with catalase, abolished the bactericidal effect. This raised possibilities that either the abrupt killing prevented accumulation of ·OH (dead cells did not generate ·OH) or that DNA degradation and killing is the actual footprint of ·OH action. In conclusion, honeys of buckwheat origin exhibited powerful, concentration-dependent bactericidal effect. The killing and DNA degradation showed a cause-effect relationship. Hydrogen peroxide was an active part of honey killing mechanism.

Brudzynski, Katrina; Abubaker, Kamal; Wang, Tony

2012-01-01

227

Development and validation of an analytical method to determine Fipronil and its degradation products in soil samples.  

PubMed

The aim of this study was to develop a methodology for identifying and quantifying Fipronil and its degradation products in soil by gas chromatography-electron capture detector previously extracted using a focused ultrasound probe. This methodology was obtaining a range of recovery between 85% and 120%, decreasing approximately solvent used time and cost, respect to other methodologies such as bath ultrasonic, solid-phase extraction, liquid-liquid extraction and soxhlet. The method was validated in fortified matrix, presented linearity in the range of 25-400 ?g kg(-1), and limit of detection for Fipronil and their products desulfinyl, sulfide and sulfone was 14.7, 9.8, 8.9 and 10.7 ?g kg(-1), respectively. This process was applied to samples of agricultural soils, where two degradation products desulfinyl and sulfone were found. PMID:22893178

Flores-Ramírez, R; Batres-Esquivel, L E; Díaz-Barriga Martínez, F; López-Acosta, I; Ortiz-Pérez, M D

2012-10-01

228

Protein quantity on the air-solid interface determines degradation rates of human growth hormone in lyophilized samples.  

PubMed

Recombinant human growth hormone (rhGH) was lyophilized with various glass-forming stabilizers, employing cycles that incorporated various freezing and annealing procedures to manipulate glass formation kinetics, associated relaxation processes, and glass-specific surface areas (SSAs). The secondary structure in the cake was monitored by infrared and in reconstituted samples by circular dichroism. The rhGH concentrations on the surface of lyophilized powders were determined from electron spectroscopy for chemical analysis. Glass transition temperature (Tg ), SSAs, and water contents were determined immediately after lyophilization. Lyophilized samples were incubated at 323 K for 16 weeks, and the resulting extents of rhGH aggregation, oxidation, and deamidation were determined after rehydration. Water contents and Tg were independent of lyophilization process parameters. Compared with samples lyophilized after rapid freezing, rhGH in samples that had been annealed in frozen solids prior to drying, or annealed in glassy solids after secondary drying retained more native-like protein secondary structure, had a smaller fraction of the protein on the surface of the cake, and exhibited lower levels of degradation during incubation. A simple kinetic model suggested that the differences in the extent of rhGH degradation during storage in the dried state between different formulations and processing methods could largely be ascribed to the associated levels of rhGH at the solid-air interface after lyophilization. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1356-1366, 2014. PMID:24623139

Xu, Yemin; Grobelny, Pawel; Von Allmen, Alexander; Knudson, Korben; Pikal, Michael; Carpenter, John F; Randolph, Theodore W

2014-05-01

229

Comparison between mitochondrial DNA sequences in low grade astrocytomas and corresponding blood samples  

PubMed Central

Background/Aims: To identify somatic mutations in the mitochondrial DNA of glioblastomas, in a previous study the displacement loops of 17 glioblastomas and corresponding blood samples were sequenced and instabilities in repeats or transitions were detected in seven tumours. This study was extended by sequencing 10 DNA samples of diffuse astrocytomas (World Health Organisation grade II) and corresponding blood samples. Methods: The 10 DNA samples of diffuse astrocytomas and corresponding blood samples were amplified and sequenced using fluorescent nucleotides. Results: No sequence differences were detected, with the exception of a quantitative shift between two genotypes heteroplasmic within the hypervariable region 2, which can be interpreted as mitotic drift. In the glioblastoma series, any particular somatic mutation was usually found in only one tumour. The only frequent alteration was coupled to a mitochondrial germline polymorphism under-represented in the low grade astrocytoma group. Moreover, a single mutation in two patients with secondary glioblastomas had already been detected in diffuse astrocytomas of these individuals. Conclusions: A lower percentage of mitochondrial DNA mutations in low grade tumours cannot be deduced from these data.

Kirches, E; Krause, G; Weis, S; Mawrin, C; Dietzmann, K

2002-01-01

230

DNA Delivery from Matrix Metalloproteinase Degradable Poly(ethylene glycol) Hydrogels to Mouse Cloned Mesenchymal Stem Cells  

PubMed Central

The ability to genetically modify mesenchymal stem cells (MSCs) seeded inside synthetic hydrogel scaffolds would offer an alternative approach to guide MSC differentiation and to study molecular pathways in three dimensions than protein delivery. In this report, we explored gene transfer to infiltrating MSCs into matrix metalloproteinase (MMP) degradable hydrogels that were loaded with DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes were encapsulated inside poly(ethylene glycol) (PEG) hydrogels crosslinked with MMP degradable peptides via Michael Addition chemistry. A large fraction of encapsulated polyplexes remained active after encapsulation (65%) and the mechanical properties of the hydrogels were unchanged by the encapsulation of the polyplexes. Cells were seeded inside the hydrogel scaffolds using two different approaches, clustered and homogeneous. The viability of MSCs was similar in hydrogels with and without polyplexes. Transgene expression was characterized with time using a secreted reporter gene and showed different profiles for clustered and homogeneously seeded cells. Clustered cells resulted in cumulative transgene expression that increased through the 21-day incubation, while homogeneously seeded cells resulted in cumulative transgene expression that plateaued after 7-days of culture. The use of hydrogel scaffolds that allow cellular infiltration to deliver DNA may result in long lasting signals in vivo, which are essential for the regeneration of functional tissues.

Lei, Yuguo; Segura, Tatiana

2008-01-01

231

DNA delivery from matrix metalloproteinase degradable poly(ethylene glycol) hydrogels to mouse cloned mesenchymal stem cells.  

PubMed

The ability to genetically modify mesenchymal stem cells (MSCs) seeded inside synthetic hydrogel scaffolds would offer an alternative approach to guide MSC differentiation and to study molecular pathways in three dimensions than protein delivery. In this report, we explored gene transfer to infiltrating MSCs into matrix metalloproteinase (MMP) degradable hydrogels that were loaded with DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes were encapsulated inside poly(ethylene glycol) (PEG) hydrogels crosslinked with MMP-degradable peptides via Michael addition chemistry. A large fraction of encapsulated polyplexes remained active after encapsulation (65%) and the mechanical properties of the hydrogels were unchanged by the encapsulation of the polyplexes. Cells were seeded inside the hydrogel scaffolds using two different approaches: clustered and homogeneous. The viability of MSCs was similar in hydrogels with and without polyplexes. Transgene expression was characterized with time using a secreted reporter gene and showed different profiles for clustered and homogeneously seeded cells. Clustered cells resulted in cumulative transgene expression that increased through the 21-day incubation, while homogeneously seeded cells resulted in cumulative transgene expression that plateaued after 7 days of culture. The use of hydrogel scaffolds that allow cellular infiltration to deliver DNA may result in long lasting signals in vivo, which are essential for the regeneration of functional tissues. PMID:18838159

Lei, Yuguo; Segura, Tatiana

2009-01-01

232

Effects of sample collection and storage conditions on DNA damage in buccal cells from agricultural workers.  

PubMed

Buccal cells are becoming a widely used tissue source for monitoring human exposure to occupational and environmental genotoxicants. A variety of methods exist for collecting buccal cells from the oral cavity, including rinsing with saline, mouthwash, or scraping the oral cavity. Buccal cells are also routinely cryopreserved with dimethyl sulfoxide (DMSO), then examined later for DNA damage by the comet assay. The effects of these different sampling procedures on the integrity of buccal cells for measuring DNA damage are unknown. This study examined the influence of the collection and cryopreservation of buccal cells on cell survival and DNA integrity. In individuals who rinsed with Hank's balanced salt solution (HBSS), the viability of leukocytes (90%) was significantly (p<0.01) greater than that of epithelial cells (12%). Similar survival rates were found for leukocytes (88%) and epithelial cells (10%) after rinsing with Listerine(®) mouthwash. However, the viability of leukocytes after cryopreservation varied significantly (p<0.01) with DMSO concentration. Cell survival was greatest at 5% DMSO. Cryopreservation also influenced the integrity of DNA in the comet assay. Although tail length and tail moment were comparable in fresh or cryopreserved samples, the average head intensity for cryopreserved samples was ?6 units lower (95% CI: 0.8-12 units lower) than for fresh samples (t(25)=-2.36, p=0.026). These studies suggest that the collection and storage of buccal samples are critical factors for the assessment of DNA damage. Moreover, leukocytes appear to be a more reliable source of human tissue for assessing DNA damage and possibly other biochemical changes. PMID:21138773

Muniz, Juan F; McCauley, Linda A; Pak, Victoria; Lasarev, Michael R; Kisby, Glen E

2011-02-28

233

Three-phase hollow fiber liquid-phase microextraction of organophosphorous nerve agent degradation products from complex samples.  

PubMed

Degradation products of chemical warfare agents are considered as important environmental and biological markers of chemical attacks. Alkyl methylphosphonic acids (AMPAs), resulting from the fast hydrolysis of nerve agents, such as sarin and soman, and the methylphosphonic acid (MPA), final degradation product of AMPAs, were determined from complex matrices by using an emergent and miniaturized extraction technique, the hollow fiber liquid-phase microextraction (HF-LPME), before their analysis by liquid chromatography coupled to mass spectrometry (LC-MS). After studying different conditions of separation in the reversed phase LC-MS analysis, the sample treatment method was set up. The three-phase HF-LPME was carried out by using a porous polypropylene (PP) hollow fiber impregnated with 1-octanol that separates the donor and acceptor aqueous media. Various extraction parameters were evaluated such as the volume of the sample, the effect of the pH and the salt addition to the sample, the pH of the acceptor phase, the extraction temperature, the stirring speed of the sample, the immersion time in the organic solvent and the time of extraction. The optimum conditions were applied to the determination of MPA and five AMPAs in real samples, such as surface waters and urine. Compounds were extracted from a 3 mL acidified sample into only 6 ?L of alkaline water without any other pretreatment of the complex matrices. Enrichment factors (EFs) higher than 170 were obtained for three less polar AMPAs. Limits of quantification (LOQs) in the 0.013-5.3 ng mL(-1) range were obtained after microextraction of AMPAs from river water and in the range of 0.056-4.8 ng mL(-1) from urine samples with RSD values between 1 and 9%. PMID:22705170

Desoubries, Charlotte; Chapuis-Hugon, Florence; Bossée, Anne; Pichon, Valérie

2012-07-01

234

Investigation of mtDNA control region sequences in an Egyptian population sample.  

PubMed

The sequences of mitochondrial DNA (mtDNA) control region were investigated in 101 unrelated individuals living in the northern region of Nile delta (Gharbia, N=55 and Kafrelsheikh, N=46). DNA was extracted from blood stained filter papers or buccal swabs. HV1, HV2 and HV3 were PCR amplified and sequenced; the resulted sequences were aligned and compared with revised Cambridge sequence (rCRS). The results revealed presence of total 93 different haplotypes, 86 of them are unique and 7 are shared haplotypes, the most common haplotype, was observed with a frequency, 2.97% of population sample. High mtDNA diversity was observed with genetic diversity and power of discrimination, 0.9982 and 0.9883, respectively. In this dataset the west Eurasian haplogroups predominated over the African haplogroups. The results would be useful for forensic examinations and human genetic studies. PMID:23910099

Elmadawy, Mostafa Ali; Nagai, Atsushi; Gomaa, Ghada M; Hegazy, Hanaa M R; Shaaban, Fawzy Eid; Bunai, Yasuo

2013-11-01

235

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

PubMed

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. PMID:22722562

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

2012-01-01

236

Granulomatous Pneumocystis carinii pneumonia: DNA amplification studies on bronchoscopic alveolar lavage samples.  

PubMed Central

Three HIV positive subjects presented with symptoms and radiographic changes suggestive of Pneumocystis carinii pneumonia. Methenamine silver staining of bronchoscopic alveolar lavage (BAL) fluid was negative (from one sample in one patient and two samples in the other two patients). Open lung biopsy was performed because of uncertain clinical progress and diagnosis; all three patients were found to have multiple pulmonary granulomata encasing numerous P carinii organisms. DNA amplification, using P carinii specific oligonucleotides, was performed on stored bronchoscopic BAL samples. P carinii specific amplification product was detected by ethidium bromide staining after electrophoretic separation on agarose gel in one case, and by the more sensitive technique of oligohybridisation in all three cases. In granulomatous P carinii pneumonia organisms are rarely identified in bronchoscopic alveolar lavage samples using histochemical staining, but are detectable by DNA amplification, although not at levels which can be readily distinguished from low, subclinical infection.

Wakefield, A E; Miller, R F; Guiver, L A; Hopkin, J M

1994-01-01

237

Optical effects module. [housing instruments used to measure degradation of optical samples from contamination during orbital operations  

NASA Technical Reports Server (NTRS)

The possible degradation of optical samples exposed to the effluent gases and particulate matter emanating from the payload of the space transportation system during orbital operations may be determined by measuring two optical parameters for five samples exposed to this environment, namely transmittance and diffuse reflectance. Any changes detected in these parameters as a function of time during the mission are then attributable to surface contamination or to increased material absorption. These basic functions are attained in the optical effects module by virtue of the following subsystems which are described: module enclosure; light source with collimator and modulator; sample wheel with holders and rotary drive; photomultipliers for radiation detection; processing and sequencing electronic circuitry; and power conditioning interfaces. The functions of these subsystems are reviewed and specified.

1978-01-01

238

Evaluation of New Preanalysis Sample Treatment Tools and DNA Isolation Protocols To Improve Bacterial Pathogen Detection in Whole Blood?  

PubMed Central

Two novel preanalysis sample treatment tools were evaluated in combination with four DNA extraction kits for the selective isolation of bacterial DNA from whole blood. The combination of performing a preanalysis sample treatment and using a larger sample volume increased the detection limit to 50 CFU per ml.

Hansen, Wendy L. J.; Bruggeman, Cathrien A.; Wolffs, Petra F. G.

2009-01-01

239

Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples  

PubMed Central

Potent PCR inhibitors in blood and soil samples can cause false negative results from PCR-based clinical and forensic tests. We show that the effect of these inhibitors is primarily upon Taq DNA polymerase, since mutational alteration of the polymerase can overcome the inhibition to the extent that no DNA purification is now required. An N-terminal deletion (Klentaq1) is some 10–100-fold inhibition resistant to whole blood compared to full-length, wild-type (w.t.) Taq, which is strongly inhibited by 0.1–1% blood. Further mutations at codon 708, both in Klentaq 1 and Taq, confer enhanced resistance to various inhibitors of PCR reactions, including whole blood, plasma, hemoglobin, lactoferrin, serum IgG, soil extracts and humic acid, as well as high concentrations of intercalating dyes. Blood PCR inhibitors can predominantly reduce the DNA extension speed of the w.t. Taq polymerase as compared to the mutant enzymes. Single-copy human genomic targets are readily amplified from whole blood or crude soil extract, without pretreatment to purify the template DNA, and the allowed increase in dye concentration overcomes fluorescence background and quenching in real-time PCR of blood.

Kermekchiev, Milko B.; Kirilova, Lyubka I.; Vail, Erika E.; Barnes, Wayne M.

2009-01-01

240

Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors.  

PubMed

The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications. PMID:23790450

Cox, Annie M; Goodwin, Kelly D

2013-08-15

241

Nonisotopic Detection and Typing of Human Papillomavirus DNA in Genital Samples by the Line Blot Assay  

PubMed Central

The line blot assay, a gene amplification method that combines PCR with nonisotopic detection of amplified DNA, was evaluated for its ability to detect human papillomavirus (HPV) DNA in genital specimens. Processed samples were amplified with biotin-labeled primers for HPV detection (primers MY09, MY11, and HMB01) and for ?-globin detection (primers PC03 and PC04). Amplified DNA products were hybridized by a reverse blot method with oligonucleotide probe mixtures fixed on a strip that allowed the identification of 27 HPV genotypes. The line blot assay was compared to a standard consensus PCR test in which HPV amplicons were detected with radiolabeled probes in a dot blot assay. Two hundred fifty-five cervicovaginal lavage specimens and cervical scrapings were tested in parallel by both PCR tests. The line blot assay consistently detected 25 copies of HPV type 18 per run. The overall positivity for the DNA of HPV types detectable by both methods was 37.7% (96 of 255 samples) by the line blot assay, whereas it was 43.5% (111 of 255 samples) by the standard consensus PCR assay. The sensitivity and specificity of the line blot assay reached 84.7% (94 of 111 samples) and 98.6% (142 of 144 samples), respectively. The agreement for HPV typing between the two PCR assays reached 83.9% (214 of 255 samples). Of the 37 samples with discrepant results, 33 (89%) were resolved by avoiding coamplification of ?-globin and modifying the amplification parameters. With these modifications, the line blot assay compared favorably to an assay that used radiolabeled probes. Its convenience allows the faster analysis of samples for large-scale epidemiological studies. Also, the increased probe spectrum in this single hybridization assay permits more complete type discrimination.

Coutlee, Francois; Gravitt, Patti; Richardson, Harriet; Hankins, Catherine; Franco, Eduardo; Lapointe, Normand; Voyer, Helene

1999-01-01

242

Detection of Chlamydia pneumoniae DNA in Buffy-Coat Samples of Patients with Abdominal Aortic Aneurysm  

Microsoft Academic Search

Recent studies have suggested that Chlamydia pneumoniae infection is a risk factor for abdominal aortic aneurysm. This study explores the presence of Chlamydia pneumoniae DNA in buffy-coat samples of control subjects and of patients with abdominal aortic aneurysm. The seroepidemiological association\\u000a between abdominal aortic aneurysm and Chlamydia pneumoniae was also investigated. Buffy-coat samples and serum specimens were obtained from 88

B. Maraha; M. den Heijer; M. Wullink; A. van der Zee; A. Bergmans; H. Verbakel; M. Kerver; S. Graafsma; S. Kranendonk; M. Peeters

2001-01-01

243

Evaluating the Use of DNA and RNA Degradation for Estimating the Post-Mortem Interval.  

National Technical Information Service (NTIS)

Estimating the post-mortem interval (PMI) is difficult due to the many factors that influence the decomposition process. Tissues such as nails, teeth and bones are more resilient to environmental factors. Measuring the rate of degradation of nucleic acids...

A. A. Vass E. Williams J. M. Curran R. I. Fleming

2013-01-01

244

Application of the iPLEX™ Gold SNP genotyping method for the analysis of Amerindian ancient DNA samples: benefits for ancient population studies.  

PubMed

Important developments in the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique have generated new perspectives regarding SNP genotyping, which are particularly promising for ancient population-based studies. The main aim of the present study was to investigate the application of a MALDI-TOF MS-based SNP genotyping technique, called iPLEX(®) Gold, to analyze Amerindian ancient DNA samples. The first objective was to test the sensitivity of the method, which is recommended for DNA quantities between 10 and 5?ng, for ancient biological samples containing DNA molecules that were degraded and present in minute quantities. The second objective was to detail the advantages of this technique for studies on ancient populations. Two multiplexes were designed, allowing the major Amerindian mitochondrial and Y haplogroups to be determined simultaneously. This analysis has never been described before. Results demonstrated the reliability and accuracy of the method; data were obtained for both mitochondrial and nuclear DNA using picogram (pg) quantities of nucleic acid. This technique has the advantages of both MS and minisequencing techniques; thus, it should be included in the protocols for future ancient DNA studies. PMID:21298665

Mendisco, Fanny; Keyser, Christine; Hollard, Clémence; Seldes, Veronica; Nielsen, Axel E; Crubézy, Eric; Ludes, Bertrand

2011-02-01

245

An evolutionarily conserved function of proliferating cell nuclear antigen for Cdt1 degradation by the Cul4-Ddb1 ubiquitin ligase in response to DNA damage.  

PubMed

The DNA replication licensing factor Cdt1 is degraded by the ubiquitin-proteasome pathway during S phase of the cell cycle, to ensure one round of DNA replication during each cell division and in response to DNA damage to halt DNA replication. Constitutive expression of Cdt1 causes DNA re-replication and is associated with the development of a subset of human non-small cell-lung carcinomas. In mammalian cells, DNA damage-induced Cdt1 degradation is catalyzed by the Cul4-Ddb1-Roc1 E3 ubiquitin ligase. We report here that overexpression of the proliferating cell nuclear antigen (PCNA) inhibitory domain from the CDK inhibitors p21 and p57, but not the CDK-cyclin inhibitory domain, blocked Cdt1 degradation in cultured mammalian cells after UV irradiation. In vivo soluble Cdt1 and PCNA co-elute by gel filtration and associate with each other physically. Silencing PCNA in cultured mammalian cells or repression of pcn1 expression in fission yeast blocked Cdt1 degradation in response to DNA damage. Unexpectedly, deletion of Ddb1 in fission yeast cells also accumulated Cdt1 in the absence of DNA damage. We suggest that the Cul4-Ddb1 ligase evolved to ubiquitinate Cdt1 during normal cell growth as well as in response to DNA damage and a separate E3 ligase, possibly SCF(Skp2), evolved to either share or take over the function of Cdt1 ubiquitination during normal cell growth and that PCNA is involved in mediating Cdt1 degradation by the Cul4-Ddb1 ligase in response to DNA damage. PMID:16407242

Hu, Jian; Xiong, Yue

2006-02-17

246

Analysis of pooled DNA samples on high density arrays without prior knowledge of differential hybridization rates  

Microsoft Academic Search

Array based DNA pooling techniques facilitate genome-wide scale genotyping of large samples. We describe a structured analysis method for pooled data using internal replication information in large scale genotyping sets. The method takes advantage of information from single nucleotide polymorphisms (SNPs) typed in parallel on a high density array to construct a test statistic with desir- able statistical properties. We

Stuart Macgregor; Peter M. Visscher; Grant Montgomery

2006-01-01

247

Evaluation of the detection of Borrelia burgdorferi DNA in urine samples by polymerase chain reaction  

Microsoft Academic Search

Summary It is difficult in some cases to identify an infection caused byBorrelia burgdorferi and to monitor the effect of therapy. Seropositivity will persist even after successful treatment and therefore may suggest ongoing infection. For direct detection ofB. burgdorferi DNA in human urine samples, the polymerase chain reaction (PCR) was evaluated. A published primer system was selected, which amplifies a

M. Maiwald; C. Stockinger; H.-G. Sonntag; D. Hassler; M. von Knebel Doeberitz

1995-01-01

248

Fecal samples as DNA source for the diagnosis of Necrotizing Hepatopancreatitis (NHP) in Penaeus vannamei broodstock.  

PubMed

Necrotizing Hepatopancreatitis (NHP) is a severe disease of cultivated penaeid shrimp caused by a pleomorphic, gram-negative, intracellular rickettsia-like bacterium. Current diagnostic methods for this disease are invasive, requiring dissection of the animal to perform histopathological analysis. In Colombia, NHP affects mainly broodstock, being a major cause of mortalities in maturation laboratories. In order to identify the presence of NHP without having to dissect the animal, we developed a PCR-based method using fecal samples as the DNA source. The DNA was extracted using a quick isolation protocol followed by amplification with primers specific for 16S ribosomal RNA gene sequences. To verify the sensitivity and specificity we analyzed samples from the same animal by PCR and in situ hybridization, and found 100% agreement. In addition, we amplified DNA extracted form paraffin blocks to confirm NHP diagnosis. PCR amplification from fecal samples and paraffin blocks yielded the expected 440 bp fragment. We conclude that PCR amplification from fecal samples is a valuable tool for the diagnosis of NHP in broodstock organisms, and that paraffin-fixed tissues can be used as a source of DNA for PCR amplification of NHP. PMID:12887257

Briñez, Boris; Aranguren, Fernando; Salazar, Marcela

2003-06-20

249

Patterning of a nanoporous membrane for multi-sample DNA extraction  

Microsoft Academic Search

A novel method for patterning a nanoporous aluminum oxide membrane was developed using SU-8 to allow for extraction of DNA from multiple samples simultaneously. To facilitate the patterning process, the spin curve for SU-8 2015 on the membrane was determined. The correlation profile of the SU-8 spin curve was similar in form to curves of SU-8 spun on silicon wafers,

Jungkyu Kim; Karl V. Voelkerding; Bruce K. Gale

2006-01-01

250

Characterization of 26 MiniSTR Loci for Improved Analysis of Degraded DNA Samples  

Microsoft Academic Search

An additional 20 novel mini-short tandem repeat (miniSTR) loci have been developed and characterized beyond the six previously developed by our laboratory for a total of 26 non-CODIS miniSTR markers. These new markers produce short PCR products in the target range of 50-150 base pairs (bp) by moving the primer sequences as close as possible—often directly next to the identified

Carolyn R. Hill; Margaret C. Kline; Michael D. Coble; John M. Butler

2008-01-01

251

Microbial degradation of nitrogen, oxygen, and sulfur heterocyclic compounds under anaerobic conditions: Studies with aquifer samples  

SciTech Connect

The potential for anaerobic biodegradation of 12 heterocyclic model compounds was studied. Nine of the model compounds were biotransformed in aquifer slurries under sulfate-reducing or methanogenic conditions. The nitrogen and oxygen heterocyclic compounds were more susceptible to anaerobic biodegradation than those compounds containing a sulfur deteroatom. In contrast, only small amounts of methane were detected in aquifer slurries amended with compounds containing an oxygen heteroatom, even though a decrease in the parent substrate concentration occurred. Pyridine, 2-picoline and 4-picoline were biotransformed within three months under sulfate-reducing conditions. However, longer incubation times were required for the degradation of these substrates in methanogenic aquifer slurries. A literature survey reveals the widespread contamination of ground waters with heterocyclic compounds from waste management practice and fossil-fuel-related industries.

Kuhn, E.P.; Suflita, J.M.

1989-01-01

252

Increased DNA amplification success of non-invasive genetic samples by successful removal of inhibitors from faecal samples collected in the field  

Microsoft Academic Search

The use of non-invasive genetic sampling (NGS) is becoming increasingly important in the study of wild animal populations.\\u000a Obtaining DNA from faecal samples is of particular interest because faeces can be collected without deploying sample capture\\u000a devices. However, PCR amplification of DNA extracted from faeces is problematic because of high concentrations of inhibitors.\\u000a Here we present a method for increasing

Louise Hebert; Safi K. Darden; Bo Vest Pedersen; Torben Dabelsteen

2011-01-01

253

Antibiotic Resistance Genes in the Bacteriophage DNA Fraction of Environmental Samples  

PubMed Central

Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, ?-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to ?-lactam antibiotics is conferred by ?-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to ?-lactam antibiotics, namely two ?-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment.

Colomer-Lluch, Marta; Jofre, Juan; Muniesa, Maite

2011-01-01

254

Detection of pyrethroid pesticides and their environmental degradation products in duplicate diet samples  

EPA Science Inventory

The abstract is for an oral presentation at the Asilomar Conference on Mass Spectrometry: Mass Spectrometry in Environmental Chemistry, Toxicology, and Health. It describes analytical method development and sample results for determination of pyrethroid pesticides and environme...

255

Discovery of rare mutations in extensively pooled DNA samples using multiple target enrichment.  

PubMed

Chemical mutagenesis is routinely used to create large numbers of rare mutations in plant and animal populations, which can be subsequently subjected to selection for beneficial traits and phenotypes that enable the characterization of gene functions. Several next-generation sequencing (NGS)-based target enrichment methods have been developed for the detection of mutations in target DNA regions. However, most of these methods aim to sequence a large number of target regions from a small number of individuals. Here, we demonstrate an effective and affordable strategy for the discovery of rare mutations in a large sodium azide-induced mutant rice population (F2 ). The integration of multiplex, semi-nested PCR combined with NGS library construction allowed for the amplification of multiple target DNA fragments for sequencing. The 8 × 8 × 8 tridimensional DNA sample pooling strategy enabled us to obtain DNA sequences of 512 individuals while only sequencing 24 samples. A stepwise filtering procedure was then elaborated to eliminate most of the false positives expected to arise through sequencing error, and the application of a simple Student's t-test against position-prone error allowed for the discovery of 16 mutations from 36 enriched targeted DNA fragments of 1024 mutagenized rice plants, all without any false calls. PMID:24602056

Chi, Xu; Zhang, Yingchun; Xue, Zheyong; Feng, Laibao; Liu, Huaqing; Wang, Feng; Qi, Xiaoquan

2014-08-01

256

Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration  

PubMed Central

HIV-1 reverse transcriptase discriminates poorly between dUTP and dTTP, and accordingly, viral DNA products become heavily uracilated when viruses infect host cells that contain high ratios of dUTP:dTTP. Uracilation of invading retroviral DNA is thought to be an innate immunity barrier to retroviral infection, but the mechanistic features of this immune pathway and the cellular fate of uracilated retroviral DNA products is not known. Here we developed a model system in which the cellular dUTP:dTTP ratio can be pharmacologically increased to favor dUTP incorporation, allowing dissection of this innate immunity pathway. When the virus-infected cells contained elevated dUTP levels, reverse transcription was found to proceed unperturbed, but integration and viral protein expression were largely blocked. Furthermore, successfully integrated proviruses lacked detectable uracil, suggesting that only nonuracilated viral DNA products were integration competent. Integration of the uracilated proviruses was restored using an isogenic cell line that had no detectable human uracil DNA glycosylase (hUNG2) activity, establishing that hUNG2 is a host restriction factor in cells that contain high dUTP. Biochemical studies in primary cells established that this immune pathway is not operative in CD4+ T cells, because these cells have high dUTPase activity (low dUTP), and only modest levels of hUNG activity. Although monocyte-derived macrophages have high dUTP levels, these cells have low hUNG activity, which may diminish the effectiveness of this restriction pathway. These findings establish the essential elements of this pathway and reconcile diverse observations in the literature.

Weil, Amy F.; Ghosh, Devlina; Zhou, Yan; Seiple, Lauren; McMahon, Moira A.; Spivak, Adam M.; Siliciano, Robert F.; Stivers, James T.

2013-01-01

257

Swabs as DNA collection devices for sampling different biological materials from different substrates.  

PubMed

Currently, there is a variety of swabs for collection of biological evidence from crime scenes, but their comparative efficiency is unknown. Here, we report the results of an investigation into the efficiency of different swab types to collect blood, saliva and touch DNA from a range of substrates. The efficiency of extracting blood and saliva from each swab type was also tested. Some swabs were significantly more effective than others for sampling biological materials from different substrates. Swabs with the highest sampling efficiency, however, often did not have the highest extraction efficiency. Observations were recorded regarding practicality of each swab in a variety of situations. Our study demonstrates that selection of sampling device impacts greatly upon successful collection and extraction of DNA. We present guidelines to assist in evaluation of swab choice. PMID:24502761

Verdon, Timothy J; Mitchell, Robert J; van Oorschot, Roland A H

2014-07-01

258

Cytomegalovirus DNA stability in EDTA Anti-Coagulated Whole Blood and Plasma Samples  

PubMed Central

Background Cytomegalovirus (CMV) DNA viral load testing is routinely performed in centers that serve patients that are immunosuppressed from organ or hematopoietic stem cell transplantation. Clinical laboratories that offer this testing often face practical concerns about the storage of these specimens to ensure accurate measurement for patient care. The studies published that look at CMV DNA stability at 4°C have done so only up to 72 hours. Objective Our objective was to determine the stability of CMV DNA in whole blood and plasma for clinical viral load testing over a 14 day period. Study Design Twenty-one plasma samples that were CMV-positive and twenty whole blood samples (including eleven CMV-negative whole blood samples spiked with CMV-positive plasma) were stored at 4°C and underwent extraction and amplification at 3 time points: Day 0, Day 7, and Day 14. Results Log10 values were calculated and t-test was performed on the values comparing Day 0 to Day 14 for plasma and whole blood. There was no statistically significant difference between Day 0 and Day 14 for both specimen types, including the CMV-negative whole blood that was spiked with CMV-positive plasma. Conclusions CMV DNA in plasma and whole blood is stable for 14 days at a temperature of 4°C.

Abdul-Ali, Deborah; Kraft, Colleen S.; Ingersoll, Jessica; Frempong, Mona

2011-01-01

259

Antibiotic Resistance Genes in the Bacteriophage DNA Fraction of Human Fecal Samples  

PubMed Central

A group of antibiotic resistance genes (ARGs) (blaTEM, blaCTX-M-1, mecA, armA, qnrA, and qnrS) were analyzed by real-time quantitative PCR (qPCR) in bacteriophage DNA isolated from feces from 80 healthy humans. Seventy-seven percent of the samples were positive in phage DNA for one or more ARGs. blaTEM, qnrA, and, blaCTX-M-1 were the most abundant, and armA, qnrS, and mecA were less prevalent. Free bacteriophages carrying ARGs may contribute to the mobilization of ARGs in intra- and extraintestinal environments.

Quiros, Pablo; Colomer-Lluch, Marta; Martinez-Castillo, Alexandre; Miro, Elisenda; Argente, Marc; Jofre, Juan; Navarro, Ferran

2014-01-01

260

Rapid extraction of PCR-competent DNA from recalcitrant environmental samples.  

PubMed

Advances in sequencing technologies have made the investigation of microbial ecology and community dynamics more tractable. The critical first step in such analyses is the efficient and representative recovery of PCR-competent DNA from complex environmental samples. All extraction protocols contain inherent biases, meaning that choice of method involves compromise between various factors, including efficiency, yield, universality, and representative extraction. Here, details are given for a routine method used in our laboratory to extract DNA from soils, sediments, biofilms, roots, and fungi. PMID:24515357

Gillings, Michael R

2014-01-01

261

Dry sampling of gas-phase isocyanates and isocyanate aerosols from thermal degradation of polyurethane.  

PubMed

The performance of a dry sampler, with an impregnated denuder in series with a glass fibre filter, using di-n-butylamine (DBA) for airborne isocyanates (200ml min(-1)) is investigated and compared with an impinger flask with a glass fibre filter in series (1 l min(-1)). An exposure chamber containing 1,6-hexamethylene diisocyanate (HDI), isophorone diisocyanate (IPDI), and 2,4- and 2,6-toluene diisocyanate (TDI) in the concentration range of 5-205 ?g m(-3) [0.7-33 p.p.b.; relative humidity (RH) 50%], generated by gas- and liquid-phase permeation, was used for the investigation. The precision for the dry sampling for five series with eight samplers were in the range of 2.0-6.1% with an average of 3.8%. During 120-min sampling (n = 4), no breakthrough was observed when analysing samplers in series. Sixty-four exposed samplers were analysed after storage for 0, 7, 14, and 21 days. No breakdown of isocyanate derivatives was observed. Twenty-eight samplers in groups of eight were collecting isocyanates during 0.5-32h. Virtually linear relationships were obtained with regard to sampling time and collected isocyanates with correlation coefficients in the range of 0.998-0.999 with the intercept close to the origin. Pre- or post-exposure to ambient air did not affect the result. Dry sampling (n = 48) with impinger-filter sampling (n = 48) of thermal decomposition product of polyurethane polymers, at RH 20, 40, 60, and 90%, was compared for 11 isocyanate compounds. The ratio between the different isocyanates collected with dry samplers and impinger-filter samplers was in the range of 0.80-1.14 for RH = 20%, 0.8-1.25 for RH = 40%, 0.76-1.4 for RH = 60%, and 0.72-3.7 for RH = 90%. Taking into account experimental errors, it seems clear that isocyanic acid DBA derivatives are found at higher levels in the dry samples compared with impinger-filter samplers at elevated humidity. The dry sampling using DBA as the reagent enables easy and robust sampling without the need of field extraction. PMID:23960047

Gylestam, Daniel; Riddar, Jakob B; Karlsson, Daniel; Dahlin, Jakob; Dalene, Marianne; Skarping, Gunnar

2014-01-01

262

Controlled degradation by ClpXP protease tunes the levels of the excision repair protein UvrA to the extent of DNA damage  

PubMed Central

Summary UV-irradiation damages DNA and activates expression of genes encoding proteins helpful for survival under DNA stress. These proteins are often deleterious in the absence of DNA damage. Here, we investigate mechanisms used to regulate the levels of DNA-repair proteins during recovery by studying control of the nucleotide excision repair (NER) protein UvrA. We show that UvrA is induced after UV-irradiation and reaches maximum levels between ~20 to 120 min post-UV. During post-UV recovery, UvrA levels decrease principally as a result of ClpXP-dependent protein degradation. The rate of UvrA degradation depends on the amount of unrepaired pyrimidine dimers present; this degradation rate is initially slow shortly after UV, but increases as damage is repaired. This increase in UvrA degradation as repair progresses is also influenced by protein-protein interactions. Genetic and in vitro experiments support the conclusion that UvrA-UvrB interactions antagonize degradation. In contrast, Mfd appears to act as an enhancer of UvrA turnover. Thus, our results reveal that a complex network of interactions contribute to tuning the level of UvrA in the cell in response to the extent of DNA damage and nicely mirror findings with excision repair proteins from eukaryotes, which are controlled by proteolysis in a similar manner.

Pruteanu, Mihaela; Baker, Tania A.

2010-01-01

263

NIJ Proposal to Enhance Methods for Studying Degraded DNA, Final Technical Report.  

National Technical Information Service (NTIS)

Quantitative PCR was used to evaluate the efficacy of sodium hypochlorite in the removal of contaminating DNA from bone surfaces. While our findings are consistent with previous studies that found sodium hypochlorite to be highly efficient ((approximately...

B. M. Kemp C. Grier C. Monroe J. E. Teisberg J. L. Barta K. Flanigan M. Winters

2013-01-01

264

How DNA is damaged by external electric fields: selective mutation vs. random degradation.  

PubMed

DNA is constantly exposed to exogenous agents that randomly damage the genetic code. However, external perturbations might also be used to induce malignant cell death if the mutation processes are controlled. The present communication reports a set of parameters allowing DNA mutation through the use of intense external electric fields. This is a step towards the design of pulsed electric field therapy for genetic diseases. PMID:24343518

Cerón-Carrasco, José Pedro; Cerezo, Javier; Jacquemin, Denis

2014-05-14

265

Ferrite supports for isolation of DNA from complex samples and polymerase chain reaction amplification.  

PubMed

The influence of cobalt ferrite particles, with non-modified or modified surface, on the course of polymerase chain reaction (PCR) was investigated. DNA isolated from bacterial cells of Bifidobacterium bifidum was used in PCR evaluation of magnetic microspheres. The presence of cobalt ferrite particles inhibits PCR amplification. The effect is not dependent on the functional groups of the modifying reagents used (none, amino, carboxyl). Amplification was improved after the magnetic separation of magnetic particles. Proposed indirect method enabled verification of the suitability of designed particles for their application in PCR assays. Magnetic particles coated with alginic acid under high PEG and sodium chloride concentration were used for the isolation of PCR-ready bacterial DNA from various dairy products. DNA was isolated from crude bacterial cell lysates without phenol extraction of samples. Bifidobacterium and Lactobacillus DNAs were identified in dairy products using PCR. PMID:16013619

Spanová, Alena; Rittich, Bohuslav; Benes, Milan J; Horák, Daniel

2005-07-01

266

Assessing the function of homologous recombination DNA repair in malignant pleural effusion (MPE) samples.  

PubMed

Background:Patients with malignant pleural effusions (MPEs) generally have advanced disease with poor survival and few therapeutic options. Cells within MPEs may be used to stratify patients for targeted therapy. Targeted therapy with poly(ADP ribose) polymerase inhibitors (PARPi) depends on identifying homologous recombination DNA repair (HRR)-defective cancer cells. We aimed to determine the feasibility of assaying HRR status in MPE cells.Methods:A total of 15 MPE samples were collected from consenting patients with non-small-cell lung cancer (NSCLC), mesothelioma and ovarian and breast cancer. Primary cultures were confirmed as epithelial by pancytokeratin, and HRR status was determined by the detection of ?H2AX and RAD51 foci following a 24-h exposure to rucaparib, by immunofluorescence microscopy. Massively parallel next-generation sequencing of DNA repair genes was performed on cultured MPE cells.Results:From 15 MPE samples, 13 cultures were successfully established, with HRR function successfully determined in 12 cultures. Four samples - three NSCLC and one mesothelioma - were HRR defective and eight samples - one NSCLC, one mesothelioma, one sarcomatoid, one breast and four ovarian cancers - were HRR functional. No mutations in DNA repair genes were associated with HRR status, but there was probable loss of heterozygosity of FANCG, RPA1 and PARP1.Conclusions:HRR function can be successfully detected in MPE cells demonstrating the potential to stratify patients for targeted therapy with PARPi. PMID:24867690

Patterson, M J; Sutton, R E; Forrest, I; Sharrock, R; Lane, M; Kaufmann, A; O'Donnell, R; Edmondson, R J; Wilson, B T; Curtin, N J

2014-07-01

267

DNA-based and culture-based characterization of a hydrocarbon-degrading consortium enriched from Arctic soil.  

PubMed

A hydrocarbon-degrading consortium was enriched from fuel-contaminated soil from the northeastern tip of Ellesmere Island (82 degrees 30'N, 62 degrees 19'W). The enrichment culture was grown on Jet A-1 fuel at 7 degrees C. Bacterial 16S RNA gene (rDNA) fragments were amplified by polymerase chain reaction (PCR) from members of the above consortium and cloned into a plasmid vector. Partial sequences (approximately 500 bp) were determined for 29 randomly selected rDNA clones. The majority of sequences were most similar to the corresponding rDNA sequences of Rhodococcus erythropolis (15 sequences), Sphingomonas spp. (six sequences), and Pseudomonas synxantha (four sequences). Amplified ribosomal DNA restriction analysis confirmed that a larger set of 50 clones had frequencies of the three phylotypes similar to those above. Phylotype-specific PCR assays were developed and validated for the above three phylotypes. The consortium was plated and grown on Jet A-1 fuel vapors, and randomly selected isolated colonies were screened with the above PCR assays. Of 17 colonies, six matched the Rhodococcus phylotype, and three matched the Pseudomonas phylotype. A representative strain of each phylotype was physiologically characterized. Both isolates grew on alkanes at low temperature and had general characteristics consistent with their respective phylotypes. During growth of the consortium, the three phylotype populations were monitored by a most probable number PCR assay. All three phylotypes were detected, but their relative abundance was not consistent with that of the phylotypes in the clone library. The relative abundance of all three phylotypes changed substantially during long-term incubation of the consortium. The DNA-based approach used identified phylotypes consistently present in the consortium, but it failed to predict the relative abundance of their populations. PMID:11822837

Thomassin-Lacroix, E J; Yu, Z; Eriksson, M; Reimer, K J; Mohn, W W

2001-12-01

268

Rapid estimation of genetic relatedness among heterogeneous populations of alfalfa by random amplification of bulked genomic DNA samples  

Microsoft Academic Search

A procedure which involves the use of RAPD markers, obtained from bulked genomic DNA samples, to estimate genetic relatedness among heterogeneous populations is demonstrated in this study. Bulked samples of genomic DNA from several alfalfa plants per population were used as templates in polymerase chain reactions with different random primers to produce RAPD patterns. The results show that the RAPD

Kangfu Yu; K. P. Pauls

1993-01-01

269

A Multiple-Tubes Approach for Accurate Genotyping of Very Small DNA Samples by Using PCR: Statistical Considerations  

Microsoft Academic Search

Summary A multiple-tubes procedure is described for using PCR to determine the genotype of a very small DNA sample. of each tube separately. The results are analyzed by a statistical procedure which determines whether a genotype can be conclusively assigned to the DNA sample. Simulation studies show that this procedure usually gives correct results even when the number of double-stranded

W. Navidi; N. Arnheim; M. S. Waterman

270

Quantitative polymerase chain reaction analysis of DNA from noninvasive samples for accurate microsatellite genotyping of wild chimpanzees ( Pan troglodytes verus )  

Microsoft Academic Search

Noninvasive samples are useful for molecular genetic analyses of wild animal populations. However, the low DNA content of such samples makes DNA amplification difficult, and there is the potential for erroneous results when one of two alleles at heterozygous microsatellite loci fails to be amplified. In this study we describe an assay designed to measure the amount of amplifiable nuclear

P. A. MORIN; K. E. CHAMBERS; C. BOESCH; L. VIGILANT

2001-01-01

271

Specific detection of DNA using quantum dots and magnetic beads for large volume samples  

SciTech Connect

Here we present a sensitive DNA detection protocol using quantum dots (QDs) and magnetic beads (MBs) for large volume samples. In this study, QDs, conjugated with streptavidin, were used to produce fluorescent signals while magnetic beads (MBs) were used to isolate and concentrate the signals. The presence of target DNAs lead to the sandwich hybridization between the functionalized QDs, the target DNAs and the MBs. In fact, the QDs-MBs complex, which is bound using the target DNA, can be isolated and then concentrated. The binding of the QDs to the surface of the MBs was confirmed by confocal microscopy and Cd elemental analysis. It was found that the fluorescent intensity was proportional to concentration of the target DNA, while the presence of noncomplementary DNA produced no significant fluorescent signal. In addition, the presence of low copies of target DNAs such as 0.5 pM in large volume samples up to 40 ml were successfully detected by using a magnet-assisted concentration protocol which consequently results in the enhancement of the sensitivity more than 100-fold.

Kim, Yeon S.; Kim, Byoung CHAN; Lee, Jin Hyung; Kim, Jungbae; Gu, Man Bock

2006-10-01

272

A simple method using PyrosequencingTM to identify de novo SNPs in pooled DNA samples  

PubMed Central

A practical way to reduce the cost of surveying single-nucleotide polymorphism (SNP) in a large number of individuals is to measure the allele frequencies in pooled DNA samples. PyrosequencingTM has been frequently used for this application because signals generated by this approach are proportional to the amount of DNA templates. The PyrosequencingTM pyrogram is determined by the dispensing order of dNTPs, which is usually designed based on the known SNPs to avoid asynchronistic extensions of heterozygous sequences. Therefore, utilizing the pyrogram signals to identify de novo SNPs in DNA pools has never been undertook. Here, in this study we developed an algorithm to address this issue. With the sequence and pyrogram of the wild-type allele known in advance, we could use the pyrogram obtained from the pooled DNA sample to predict the sequence of the unknown mutant allele (de novo SNP) and estimate its allele frequency. Both computational simulation and experimental PyrosequencingTM test results suggested that our method performs well. The web interface of our method is available at http://life.nctu.edu.tw/?yslin/PSM/.

Lin, Yeong-Shin; Liu, Fu-Guo Robert; Wang, Tzi-Yuan; Pan, Cheng-Tsung; Chang, Wei-Ting; Li, Wen-Hsiung

2011-01-01

273

Automated digital microfluidic sample preparation for next-generation DNA sequencing.  

PubMed

Next-generation sequencing (NGS) technology is a promising tool for identifying and characterizing unknown pathogens, but its usefulness in time-critical biodefense and public health applications is currently limited by the lack of fast, efficient, and reliable automated DNA sample preparation methods. To address this limitation, we are developing a digital microfluidic (DMF) platform to function as a fluid distribution hub, enabling the integration of multiple subsystem modules into an automated NGS library sample preparation system. A novel capillary interface enables highly repeatable transfer of liquid between the DMF device and the external fluidic modules, allowing both continuous-flow and droplet-based sample manipulations to be performed in one integrated system. Here, we highlight the utility of the DMF hub platform and capillary interface for automating two key operations in the NGS sample preparation workflow. Using an in-line contactless conductivity detector in conjunction with the capillary interface, we demonstrate closed-loop automated fraction collection of target analytes from a continuous-flow sample stream into droplets on the DMF device. Buffer exchange and sample cleanup, the most repeated steps in NGS library preparation, are also demonstrated on the DMF platform using a magnetic bead assay and achieving an average DNA recovery efficiency of 80%±4.8%. PMID:22093297

Kim, Hanyoup; Bartsch, Michael S; Renzi, Ronald F; He, Jim; Van de Vreugde, James L; Claudnic, Mark R; Patel, Kamlesh D

2011-12-01

274

Taxonomic characterization of two rubber degrading bacteria belonging to the species Gordonia polyisoprenivorans and analysis of hyper variable regions of 16S rDNA sequences.  

PubMed

Two cis-1,4-polyisoprene (isoprene rubber) degrading bacteria, strains VH2 and Y2K, were identified as strains of the species Gordonia polyisoprenivorans belonging to the Corynebacterineae, a suborder of the order Actinomycetales. Both showed characteristic growth and degradation of isoprene rubber as described previously for the type strain of G. polyisoprenivorans Kd2 (DSM 44302(T)). For strain VH2 the chemotaxonomic properties were investigated, and DNA-DNA hybridization experiments with the type strain revealed the affiliation to the species G. polyisoprenivorans. The comparison of the 16S rDNA sequences, and especially hyper variable regions of these, led to the classification of strain Y2K to the same species. At present, the species G. polyisoprenivorans comprises three different isolates which share the ability to degrade isoprene rubber potently but which were obtained from different geographic regions. PMID:11750816

Arenskötter, M; Baumeister, D; Berekaa, M M; Pötter, G; Kroppenstedt, R M; Linos, A; Steinbüchel, A

2001-12-18

275

CDC-48/p97 Coordinates CDT-1 Degradation with GINS Chromatin Dissociation to Ensure Faithful DNA Replication  

PubMed Central

SUMMARY Faithful transmission of genomic information requires tight spatiotemporal regulation of DNA replication factors. In the licensing step of DNA replication CDT-1 is loaded onto chromatin to subsequently promote the recruitment of additional replication factors including CDC-45 and GINS. During the elongation step the CDC-45/GINS complex moves with the replication fork, however it is largely unknown how its chromatin association is regulated. Here, we show that the chaperone-like ATPase CDC-48/p97 coordinates degradation of CDT-1 with release of the CDC-45/GINS complex. C. elegans embryos lacking CDC-48 or its cofactors UFD-1/NPL-4 accumulate CDT-1 on mitotic chromatin, indicating a critical role of CDC-48 in CDT-1 turnover. Strikingly, CDC-48UFD-1/NPL-4 deficient embryos show persistent chromatin association of CDC-45/GINS, which is a consequence of CDT-1 stabilization. Moreover, our data confirmed a similar regulation in Xenopus egg extracts, emphasizing a conserved coordination of licensing and elongation events during eukaryotic DNA replication by CDC-48/p97.

Franz, Andre; Orth, Michael; Pirson, Paul A.; Sonneville, Remi; Blow, J. Julian; Gartner, Anton; Stemmann, Olaf; Hoppe, Thorsten

2012-01-01

276

Detection of Cryptococcus neoformans DNA in Tissue Samples by Nested and Real-Time PCR Assays  

Microsoft Academic Search

mice treated with different azoles were examined. A fungal burden of 3 10 1 to 2.9 10 4 CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity

Ralf Bialek; Michael Weiss; Kubrom Bekure-Nemariam; Laura K. Najvar; Maria B. Alberdi; John R. Graybill; Udo Reischl

2002-01-01

277

Selection of indicator bacteria based on screening of 16S rDNA metagenomic library from a two-stage anoxic–oxic bioreactor system degrading azo dyes  

Microsoft Academic Search

Dye degradation has gained attention of late due to indiscriminate disposal from user industries. Enhancing efficiency of biological treatment provides a cheaper alternative vis-à-vis other advanced technologies. Dye molecules are metabolized biologically via anoxic and oxic treatments. In this study, bacterial community surviving on dye effluent working in anoxic–oxic bioreactor was analyzed using 16S rDNA approach. Azo-dye decolorizing and degrading

Nishant Dafale; Leena Agrawal; Atya Kapley; Sudhir Meshram; Hemant Purohit; Satish Wate

2010-01-01

278

Release of Free DNA by Membrane-Impaired Bacterial Aerosols Due to Aerosolization and Air Sampling  

PubMed Central

We report here that stress experienced by bacteria due to aerosolization and air sampling can result in severe membrane impairment, leading to the release of DNA as free molecules. Escherichia coli and Bacillus atrophaeus bacteria were aerosolized and then either collected directly into liquid or collected using other collection media and then transferred into liquid. The amount of DNA released was quantified as the cell membrane damage index (ID), i.e., the number of 16S rRNA gene copies in the supernatant liquid relative to the total number in the bioaerosol sample. During aerosolization by a Collison nebulizer, the ID of E. coli and B. atrophaeus in the nebulizer suspension gradually increased during 60 min of continuous aerosolization. We found that the ID of bacteria during aerosolization was statistically significantly affected by the material of the Collison jar (glass > polycarbonate; P < 0.001) and by the bacterial species (E. coli > B. atrophaeus; P < 0.001). When E. coli was collected for 5 min by filtration, impaction, and impingement, its ID values were within the following ranges: 0.051 to 0.085, 0.16 to 0.37, and 0.068 to 0.23, respectively; when it was collected by electrostatic precipitation, the ID values (0.011 to 0.034) were significantly lower (P < 0.05) than those with other sampling methods. Air samples collected inside an equine facility for 2 h by filtration and impingement exhibited ID values in the range of 0.30 to 0.54. The data indicate that the amount of cell damage during bioaerosol sampling and the resulting release of DNA can be substantial and that this should be taken into account when analyzing bioaerosol samples.

Zhen, Huajun; Han, Taewon; Fennell, Donna E.

2013-01-01

279

Release of free DNA by membrane-impaired bacterial aerosols due to aerosolization and air sampling.  

PubMed

We report here that stress experienced by bacteria due to aerosolization and air sampling can result in severe membrane impairment, leading to the release of DNA as free molecules. Escherichia coli and Bacillus atrophaeus bacteria were aerosolized and then either collected directly into liquid or collected using other collection media and then transferred into liquid. The amount of DNA released was quantified as the cell membrane damage index (ID), i.e., the number of 16S rRNA gene copies in the supernatant liquid relative to the total number in the bioaerosol sample. During aerosolization by a Collison nebulizer, the ID of E. coli and B. atrophaeus in the nebulizer suspension gradually increased during 60 min of continuous aerosolization. We found that the ID of bacteria during aerosolization was statistically significantly affected by the material of the Collison jar (glass > polycarbonate; P < 0.001) and by the bacterial species (E. coli > B. atrophaeus; P < 0.001). When E. coli was collected for 5 min by filtration, impaction, and impingement, its ID values were within the following ranges: 0.051 to 0.085, 0.16 to 0.37, and 0.068 to 0.23, respectively; when it was collected by electrostatic precipitation, the ID values (0.011 to 0.034) were significantly lower (P < 0.05) than those with other sampling methods. Air samples collected inside an equine facility for 2 h by filtration and impingement exhibited ID values in the range of 0.30 to 0.54. The data indicate that the amount of cell damage during bioaerosol sampling and the resulting release of DNA can be substantial and that this should be taken into account when analyzing bioaerosol samples. PMID:24096426

Zhen, Huajun; Han, Taewon; Fennell, Donna E; Mainelis, Gediminas

2013-12-01

280

Abundance of DNA adducts of methyleugenol, a rodent hepatocarcinogen, in human liver samples.  

PubMed

Methyleugenol is a genotoxic carcinogen in mice and rats, the liver being the primary target tissue. Methyleugenol occurs in fennel and many herbs and spices. Furthermore, methyleugenol-containing plant extracts and chemically prepared methyleugenol are used as flavoring agents. We analyzed surgical human liver samples from 30 subjects for the presence of DNA adducts originating from methyleugenol using isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Twenty-nine samples unambiguously contained the N (2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine adduct. A second adduct, N (6)-(trans-methylisoeugenol-3'-yl)-2'-deoxyadenosine, was also found in most samples, but at much lower levels, in agreement with the results from experimental models. The maximal and median levels of both adducts combined were 37 and 13 per 10(8) nucleosides (corresponding to 4700 and 1700, respectively, adducts per diploid genome). This is the first demonstration of DNA adducts formed by a xenobiotic in human liver using UPLC-MS/MS, the most reliable method available. It has been estimated for diverse rat and mouse hepatocarcinogens that 50-5500 adducts per 10(8) nucleosides are present after repeated treatment at the TD50 (daily dose that halves the probability to stay tumor-free in long-term studies). We conclude that the exposure to methyleugenol leads to substantial levels of hepatic DNA adducts and, therefore, may pose a significant carcinogenic risk. PMID:23334163

Herrmann, Kristin; Schumacher, Fabian; Engst, Wolfram; Appel, Klaus E; Klein, Kathrin; Zanger, Ulrich M; Glatt, Hansruedi

2013-05-01

281

Comparison of DNA Extraction Methods from Small Samples of Newborn Screening Cards Suitable for Retrospective Perinatal Viral Research  

PubMed Central

Reliable detection of viral DNA in stored newborn screening cards (NSC) would give important insight into possible silent infection during pregnancy and around birth. We sought a DNA extraction method with sufficient sensitivity to detect low copy numbers of viral DNA from small punch samples of NSC. Blank NSC were spotted with seronegative EDTA-blood and seropositive EBV EDTA-blood. DNA was extracted with commercial and noncommercial DNA extraction methods and quantified on a spectrofluorometer using a PicoGreen dsDNA quantification kit. Serial dilutions of purified viral DNA controls determined the sensitivity of the amplification protocol, and seropositive EBV EDTA-blood amplified by nested PCR (nPCR) validated the DNA extraction methods. There were considerable differences between the commercial and noncommercial DNA extraction methods (P=0.014; P=0.016). Commercial kits compared favorably, but the QIamp DNA micro kit with an added forensic filter step was marginally more sensitive. The mean DNA yield from this method was 3 ng/?l. The limit of detection was 10 viral genome copies in a 50-?l reaction. EBV nPCR detection in neat and 1:10 diluted DNA extracts could be replicated reliably. We conclude that the QIamp Micro DNA extraction method with the added forensic spin-filter step was suitable for retrospective DNA viral assays from NSC.

McMichael, Gai L.; Highet, Amanda R.; Gibson, Catherine S.; Goldwater, Paul N.; O'Callaghan, Michael E.; Alvino, Emily R.; MacLennan, Alastair H.

2011-01-01

282

Simplified method for DNA and protein staining of human hematopoietic cell samples. [Cell flow systems  

SciTech Connect

A rapid reproducible method yielding high resolution analysis of DNA and protein in human hematopoietic cell samples has been developed by modification of the propidium iodide and fluorescein isothiocyanate procedure. Cell staining involves sequential addition of each reagent (RNase, fluorescein isothiocyanate and propidium iodide) to ethanol-fixed cells and requires no centrifugation steps. Stained cells are analyzed in the reagent solutions. Analysis of bone marrow samples from multiple myeloma patients showed mixed normal and aneuploid populations with a major portion of the aneuploid cells having a significantly higher protein content. This approach permitted differential cell cycle analysis of normal and the aneuploid populations.

Crissman, H.A. (Los Alamos National Lab., NM); Egmond, J.V.; Holdrinet, R.S.; Pennings, A.; Haanen, C.

1981-01-01

283

A mathematical model of DNA degradation: Possible role of magnetic nanoparticles  

Microsoft Academic Search

A mathematical model of genome degradation is proposed that takes into\\u000aaccount a variable rate of mutation and increasing number of cells in a\\u000adeveloping human organism. The model explains known properties of cancer\\u000adevelopment, in particular, a synergism between different mutagens and an\\u000aincreased probability of cancer in the early years of life. An iteration\\u000aequation is suggested that

V. N. Binhi

2007-01-01

284

High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil  

PubMed Central

Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88–100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200 bp.

Lin, Jianghai; Kennedy, Stephen H.; Svarovsky, Therese; Rogers, Jeffrey; Kemnitz, Joseph W.; Xu, Anlong; Zondervan, Krina T.

2009-01-01

285

High-quality genomic DNA extraction from formalin-fixed and paraffin-embedded samples deparaffinized using mineral oil.  

PubMed

Extracting DNA from formalin-fixed and paraffin-embedded (FFPE) tissue remains a challenge, despite numerous attempts to develop a more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low. We extracted DNA from 140 long-term archived FFPE samples using a simple but effective deparaffinization method, removing the wax with mineral oil, and a commercially available DNA extraction kit. DNA quality was subsequently tested in a genotyping experiment with 14 microsatellite markers. High-quality DNA was obtained with a mean PCR success rate of 97% (range: 88-100%) across markers. The results suggested that DNA extracted using this novel method is likely to be suitable for genetic studies involving DNA fragments <200 bp. PMID:19698695

Lin, Jianghai; Kennedy, Stephen H; Svarovsky, Therese; Rogers, Jeffrey; Kemnitz, Joseph W; Xu, Anlong; Zondervan, Krina T

2009-12-15

286

Reduction of stutter ratios in short tandem repeat loci typing of low copy number DNA samples.  

PubMed

Increased height of stutter peaks is a phenomenon with low copy number (LCN) short tandem repeat (STR) typing that can impact interpretation. An alternative strategy of lowering the annealing/extension temperature (LT) at 56 °C was designed to attempt to decrease the heights of stutter peaks. STR typing results were generated in terms of stutter ratios using LT-PCR conditions and compared with data obtained using standard (STD) PCR conditions. DNA samples ranging from 100 to 25 pg were amplified using reagents contained in the AmpF?STR Identifiler PCR Amplification or AmpF?STR Identifiler Plus PCR Amplification kits with 32 or 34 PCR cycles. Stutter ratios decreased by an average of 14.7%, 14.9% and 18.1% at 100, 50 and 25 pg of template DNA under LT conditions compared with those of STD conditions in the Identifiler Kit amplified samples. The LT conditions also decreased average stutter ratios by 13.3% compared with those of STD conditions in the Identifiler Plus Kit amplified samples. The overall PCR efficiency obtained with STD and LT conditions with the two STR kits was comparable in terms of the number of detected alleles, peak heights and peak height ratios. These results support the hypothesis that a lower temperature annealing/extension step reduces the likelihood of slippage during PCR by enhancing the stability of the DNA polymerase/template DNA complex or the stability of the generated duplex than the conditions of the standard extension step. This stability in turn would result in lower stutter ratios. PMID:24315611

Seo, Seung Bum; Ge, Jianye; King, Jonathan L; Budowle, Bruce

2014-01-01

287

DNA Damage Focus Analysis in Blood Samples of Minipigs Reveals Acute Partial Body Irradiation  

PubMed Central

Radiation accidents frequently involve acute high dose partial body irradiation leading to victims with radiation sickness and cutaneous radiation syndrome that implements radiation-induced cell death. Cells that are not lethally hit seek to repair ionizing radiation (IR) induced damage, albeit at the expense of an increased risk of mutation and tumor formation due to misrepair of IR-induced DNA double strand breaks (DSBs). The response to DNA damage includes phosphorylation of histone H2AX in the vicinity of DSBs, creating foci in the nucleus whose enumeration can serve as a radiation biodosimeter. Here, we investigated ?H2AX and DNA repair foci in peripheral blood lymphocytes of Göttingen minipigs that experienced acute partial body irradiation (PBI) with 49 Gy (±6%) Co-60 ?-rays of the upper lumbar region. Blood samples taken 4, 24 and 168 hours post PBI were subjected to ?-H2AX, 53BP1 and MRE11 focus enumeration. Peripheral blood lymphocytes (PBL) of 49 Gy partial body irradiated minipigs were found to display 1–8 DNA damage foci/cell. These PBL values significantly deceed the high foci numbers observed in keratinocyte nuclei of the directly ?-irradiated minipig skin regions, indicating a limited resident time of PBL in the exposed tissue volume. Nonetheless, PBL samples obtained 4 h post IR in average contained 2.2% of cells displaying a pan-?H2AX signal, suggesting that these received a higher IR dose. Moreover, dispersion analysis indicated partial body irradiation for all 13 minipigs at 4 h post IR. While dose reconstruction using ?H2AX DNA repair foci in lymphocytes after in vivo PBI represents a challenge, the DNA damage focus assay may serve as a rapid, first line indicator of radiation exposure. The occurrence of PBLs with pan-?H2AX staining and of cells with relatively high foci numbers that skew a Poisson distribution may be taken as indicator of acute high dose partial body irradiation, particularly when samples are available early after IR exposure.

Lamkowski, Andreas; Forcheron, Fabien; Agay, Diane; Ahmed, Emad A.; Drouet, Michel; Meineke, Viktor; Scherthan, Harry

2014-01-01

288

DNA damage focus analysis in blood samples of minipigs reveals acute partial body irradiation.  

PubMed

Radiation accidents frequently involve acute high dose partial body irradiation leading to victims with radiation sickness and cutaneous radiation syndrome that implements radiation-induced cell death. Cells that are not lethally hit seek to repair ionizing radiation (IR) induced damage, albeit at the expense of an increased risk of mutation and tumor formation due to misrepair of IR-induced DNA double strand breaks (DSBs). The response to DNA damage includes phosphorylation of histone H2AX in the vicinity of DSBs, creating foci in the nucleus whose enumeration can serve as a radiation biodosimeter. Here, we investigated ?H2AX and DNA repair foci in peripheral blood lymphocytes of Göttingen minipigs that experienced acute partial body irradiation (PBI) with 49 Gy (± 6%) Co-60 ?-rays of the upper lumbar region. Blood samples taken 4, 24 and 168 hours post PBI were subjected to ?-H2AX, 53BP1 and MRE11 focus enumeration. Peripheral blood lymphocytes (PBL) of 49 Gy partial body irradiated minipigs were found to display 1-8 DNA damage foci/cell. These PBL values significantly deceed the high foci numbers observed in keratinocyte nuclei of the directly ?-irradiated minipig skin regions, indicating a limited resident time of PBL in the exposed tissue volume. Nonetheless, PBL samples obtained 4 h post IR in average contained 2.2% of cells displaying a pan-?H2AX signal, suggesting that these received a higher IR dose. Moreover, dispersion analysis indicated partial body irradiation for all 13 minipigs at 4 h post IR. While dose reconstruction using ?H2AX DNA repair foci in lymphocytes after in vivo PBI represents a challenge, the DNA damage focus assay may serve as a rapid, first line indicator of radiation exposure. The occurrence of PBLs with pan-?H2AX staining and of cells with relatively high foci numbers that skew a Poisson distribution may be taken as indicator of acute high dose partial body irradiation, particularly when samples are available early after IR exposure. PMID:24498326

Lamkowski, Andreas; Forcheron, Fabien; Agay, Diane; Ahmed, Emad A; Drouet, Michel; Meineke, Viktor; Scherthan, Harry

2014-01-01

289

Development of a High-Throughput Quantitative Assay for Detecting Herpes Simplex Virus DNA in Clinical Samples  

Microsoft Academic Search

We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 108 copies of HSV DNA\\/20 m lo f sample. Among duplicate samples in reproducibility runs, the assay showed

ALEXANDER J. RYNCARZ; JAMES GODDARD; ANNA WALD; MEEI-LI HUANG; BERNARD ROIZMAN; LAWRENCE COREY

1999-01-01

290

Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes.  

PubMed Central

The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10(4) physical particles of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hybridization stringency, 32P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments. Images

Jiang, X; Estes, M K; Metcalf, T G; Melnick, J L

1986-01-01

291

A DNA pooling based system to detect Escherichia coli virulence factors in fecal and wastewater samples  

PubMed Central

The availability of a useful tool for simple and timely detection of the most important virulent varieties of Escherichia coli is indispensable. To this end, bacterial DNA pools which had previously been categorized were obtained from isolated colonies as well as selected in terms of utilized phenotype; the pools were assessed by two PCR Multiplex for the detection of virulent E. coli eaeA, bfpA, stx1, stx2, ipaH, ST, LT, and aatA genes, with the 16S gene used as DNA control. The system was validated with 66 fecal samples and 44 wastewater samples. At least one positive isolate was detected by a virulent gene among the 20 that were screened. The analysis of fecal samples from children younger than 6 years of age detected frequencies of 25% LT positive strains, 8.3% eae, 8.3% bfpA, 16.7% ipaH, as well as 12.5 % aatA and ST. On the other hand, wastewater samples revealed frequencies of 25.7% eaeA positive, 30.3% stx1, 15.1% LT and 19.7% aatA. This study is an initial step toward carrying out epidemiological field research that will reveal the presence of these bacterial varieties.

Luz Maria Chacon, J; Lizeth Taylor, C; Carmen Valiente, A; Irene Alvarado, P; Ximena Cortes, B

2012-01-01

292

The 2000–2001 GEP–ISFG Collaborative Exercise on mtDNA: assessing the cause of unsuccessful mtDNA PCR amplification of hair shaft samples  

Microsoft Academic Search

We report the results of Spanish and Portuguese working group (GEP) of International Society of Forensic Genetics (ISFG) Collaborative Exercise 2001–2002 on mitochondrial DNA (mtDNA) analysis. 64 laboratories from Spain, Portugal and several Latin-American countries participated in this quality control exercise. Five samples were sent to the participating laboratories, four blood stains (M1–M4) and a sample (M5) consisting of two

Lourdes Prieto; Marta Montesino; Antonio Salas; Antonio Alonso; Cristina Albarrán; Sara Álvarez; Manuel Crespillo; Ana M Di Lonardo; Christian Doutremepuich; Isabel Fernández-Fernández; Alberto González de la Vega; Leonor Gusmão; Carlos M López; Manolo López-Soto; José A Lorente; Marcelo Malaghini; Carlos A Mart??nez; Nidia M Modesti; Ana M Palacio; Manuel Paredes; Sergio D. J Pena; Anna Pérez-Lezaun; José J Pestano; Jorge Puente; Andrea Sala; M Vide; Mart??n R Whittle; Juan J Yunis; Josefina Gómez

2003-01-01

293

Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for DNA sequencing  

SciTech Connect

The purpose of this research is to develop a multiplexed sample processing system in conjunction with multiplexed capillary electrophoresis for high-throughput DNA sequencing. The concept from DNA template to called bases was first demonstrated with a manually operated single capillary system. Later, an automated microfluidic system with 8 channels based on the same principle was successfully constructed. The instrument automatically processes 8 templates through reaction, purification, denaturation, pre-concentration, injection, separation and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were implemented to manage flow direction and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an 8-capillary array in a hot air thermal cycler. Subsequently, the sequencing ladders are directly loaded into a corresponding size-exclusion chromatographic column operated at {approximately} 60 C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at {approximately} 70 C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. DNA sequencing data from this system allow base calling up to 460 bases with accuracy of 98%.

Tan, H.

1999-03-31

294

WIP1, a homeostatic regulator of the DNA damage response, is targeted by HIPK2 for phosphorylation and degradation.  

PubMed

WIP1 (wild-type p53-induced phosphatase 1) functions as a homeostatic regulator of the ataxia telangiectasia mutated (ATM)-mediated signaling pathway in response to ionizing radiation (IR). Here we identify homeodomain-interacting protein kinase 2 (HIPK2) as a protein kinase that targets WIP1 for phosphorylation and proteasomal degradation. In unstressed cells, WIP1 is constitutively phosphorylated by HIPK2 and maintained at a low level by proteasomal degradation. In response to IR, ATM-dependent AMPK?2-mediated HIPK2 phosphorylation promotes inhibition of WIP1 phosphorylation through dissociation of WIP1 from HIPK2, followed by stabilization of WIP1 for termination of the ATM-mediated double-strand break (DSB) signaling cascade. Notably, HIPK2 depletion impairs IR-induced ?-H2AX foci formation, cell-cycle checkpoint activation, and DNA repair signaling, and the survival rate of hipk2+/- mice upon ?-irradiation is markedly reduced compared to wild-type mice. Taken together, HIPK2 plays a critical role in the initiation of DSB repair signaling by controlling WIP1 levels in response to IR. PMID:23871434

Choi, Dong Wook; Na, Wooju; Kabir, Mohammad Humayun; Yi, Eunbi; Kwon, Seonjeong; Yeom, Jeonghun; Ahn, Jang-Won; Choi, Hee-Hyun; Lee, Youngha; Seo, Kyoung Wan; Shin, Min Kyoo; Park, Se-Ho; Yoo, Hae Yong; Isono, Kyo-Ichi; Koseki, Haruhiko; Kim, Seong-Tae; Lee, Cheolju; Kwon, Yunhee Kim; Choi, Cheol Yong

2013-08-01

295

The Vif Protein of HIV Triggers Degradation of the Human Antiretroviral DNA Deaminase APOBEC3G  

Microsoft Academic Search

APOBEC3G is a human cellular enzyme that is incorporated into retroviral particles and acts to restrict retroviral replication in infected cells by deaminating dC to dU in the first (minus)-strand cDNA replication intermediate [1–5]. HIV, however, encodes a protein (virion infectivity factor, Vif [6, 7]), which overcomes APOBEC3G-mediated restriction but by an unknown mechanism. Here, we show that Vif triggers

Silvestro G Conticello; Reuben S Harris; Michael S Neuberger

2003-01-01

296

Reverse Sample Genome Probing, a New Technique for Identification of Bacteria in Environmental Samples by DNA Hybridization, and Its Application to the Identification of Sulfate-Reducing Bacteria in Oil Field Samples  

PubMed Central

A novel method for the identification of bacteria in environmental samples by DNA hybridization is presented. It is based on the fact that, even within a genus, the genomes of different bacteria may have little overall sequence homology. This allows the use of the labeled genomic DNA of a given bacterium (referred to as a “standard”) to probe for its presence and that of bacteria with highly homologous genomes in total DNA obtained from an environmental sample. Alternatively, total DNA extracted from the sample can be labeled and used to probe filters on which denatured chromosomal DNA from relevant bacterial standards has been spotted. The latter technique is referred to as reverse sample genome probing, since it is the reverse of the usual practice of deriving probes from reference bacteria for analyzing a DNA sample. Reverse sample genome probing allows identification of bacteria in a sample in a single step once a master filter with suitable standards has been developed. Application of reverse sample genome probing to the identification of sulfate-reducing bacteria in 31 samples obtained primarily from oil fields in the province of Alberta has indicated that there are at least 20 genotypically different sulfate-reducing bacteria in these samples. Images

Voordouw, Gerrit; Voordouw, Johanna K.; Karkhoff-Schweizer, Roxann R.; Fedorak, Phillip M.; Westlake, Donald W. S.

1991-01-01

297

Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil.  

PubMed

Efficient, nonselective methods to obtain DNA from the environment are needed for rapid and thorough analysis of introduced microorganisms in environmental samples and for analysis of microbial community diversity in soil. A small-scale procedure to rapidly extract and purify DNA from soils was developed for in-the-field use. Amounts of DNA released from bacterial vegetative cells, bacterial endospores, and fungal conidia were compared by using hot-detergent treatment, freeze-thaw cycles, and bead mill homogenization. Combining a hot-detergent treatment with bead mill homogenization gave the highest DNA yields from all three microbial cell types and provided DNA from the broadest range of microbial groups in a natural soil community. Only the bead mill homogenization step was effective for DNA extraction from Bacillus globigii (B. subtilis subsp. niger) endospores or Fusarium moniliforme conidia. The hot-detergent-bead mill procedure was simplified and miniaturized. By using this procedure and small-scale, field-adapted purification and quantification procedures, DNA was prepared from four different soils seeded with Pseudomonas putida cells or B. globigii spores. In a New Mexico soil, seeded bacterial targets were detected with the same sensitivity as when assaying pure bacterial DNA (2 to 20 target gene copies in a PCR mixture). The detection limit of P. putida cells and B. globigii spores in different soils was affected by the amount of background DNA in the soil samples, the physical condition of the DNA, and the amount of DNA template used in the PCR. PMID:9647816

Kuske; Banton; Adorada; Stark; Hill; Jackson

1998-07-01

298

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

PubMed

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. PMID:22310206

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

2012-09-01

299

Genotyping whole-genome-amplified DNA from 3- to 25-year-old neonatal dried blood spot samples with reference to fresh genomic DNA.  

PubMed

Stored surplus of dried blood spot (DBS) samples from neonatal screening programs constitute a vast potential for large genetic epidemiological studies. However, age of the samples and the small amounts of DNA available may limit their usage. In this study we validate genotyping accuracy and efficiency of whole-genome-amplified DNA (wgaDNA) obtained from stored DBS samples, with reference to fresh genomic DNA from the same individuals. DBS samples from 29 volunteers, stored for up to 25 years, in the Danish Neonatal Screening Biobank were included and three DNA extraction methods, each using one 3.2 mm disk, were evaluated. Four whole-genome amplification kits, and one re-amplification kit, were used. Thirty-one SNPs were genotyped using the Sequenom platform and the wgaDNA samples calls were compared with their references for accuracy and efficiency evaluation. The genotype calls done blinded by the user had in many setups a 100% call- and concordance rate. Our results showed that genotyping performance is dependent on the combination of extraction procedure and amplification method, whereas years of storage did not seem to influence in this study. Based on these results we conclude that DBS samples should be considered a reliable and potential resource for future genotyping studies. PMID:19639574

Hollegaard, Mads Vilhelm; Thorsen, Poul; Norgaard-Pedersen, Bent; Hougaard, David Michael

2009-07-01

300

Mendelian breeding units versus standard sampling strategies: Mitochondrial DNA variation in southwest Sardinia.  

PubMed

We report a sampling strategy based on Mendelian Breeding Units (MBUs), representing an interbreeding group of individuals sharing a common gene pool. The identification of MBUs is crucial for case-control experimental design in association studies. The aim of this work was to evaluate the possible existence of bias in terms of genetic variability and haplogroup frequencies in the MBU sample, due to severe sample selection. In order to reach this goal, the MBU sampling strategy was compared to a standard selection of individuals according to their surname and place of birth. We analysed mitochondrial DNA variation (first hypervariable segment and coding region) in unrelated healthy subjects from two different areas of Sardinia: the area around the town of Cabras and the western Campidano area. No statistically significant differences were observed when the two sampling methods were compared, indicating that the stringent sample selection needed to establish a MBU does not alter original genetic variability and haplogroup distribution. Therefore, the MBU sampling strategy can be considered a useful tool in association studies of complex traits. PMID:21734814

Sanna, Daria; Pala, Maria; Cossu, Piero; Dedola, Gian Luca; Melis, Sonia; Fresu, Giovanni; Morelli, Laura; Obinu, Domenica; Tonolo, Giancarlo; Secchi, Giannina; Triunfo, Riccardo; Lorenz, Joseph G; Scheinfeldt, Laura; Torroni, Antonio; Robledo, Renato; Francalacci, Paolo

2011-04-01

301

High-Throughput SNP Allele-Frequency Determination in Pooled DNA Samples by Kinetic PCR  

PubMed Central

We have developed an accurate, yet inexpensive and high-throughput, method for determining the allele frequency of biallelic polymorphisms in pools of DNA samples. The assay combines kinetic (real-time quantitative) PCR with allele-specific amplification and requires no post-PCR processing. The relative amounts of each allele in a sample are quantified. This is performed by dividing equal aliquots of the pooled DNA between two separate PCR reactions, each of which contains a primer pair specific to one or the other allelic SNP variant. For pools with equal amounts of the two alleles, the two amplifications should reach a detectable level of fluorescence at the same cycle number. For pools that contain unequal ratios of the two alleles, the difference in cycle number between the two amplification reactions can be used to calculate the relative allele amounts. We demonstrate the accuracy and reliability of the assay on samples with known predetermined SNP allele frequencies from 5% to 95%, including pools of both human and mouse DNAs using eight different SNPs altogether. The accuracy of measuring known allele frequencies is very high, with the strength of correlation between measured and known frequencies having an r2?=?0.997. The loss of sensitivity as a result of measurement error is typically minimal, compared with that due to sampling error alone, for population samples up to 1000. We believe that by providing a means for SNP genotyping up to thousands of samples simultaneously, inexpensively, and reproducibly, this method is a powerful strategy for detecting meaningful polymorphic differences in candidate gene association studies and genome-wide linkage disequilibrium scans.

Germer, S?ren; Holland, Michael J.; Higuchi, Russell

2000-01-01

302

Increased sensitivity for determination of polycyclic aromatic hydrocarbon-DNA adducts in human DNA samples by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA)  

SciTech Connect

A competitive enzyme-linked immunosorbent assay (ELISA), the most frequently used immunoassay for the determination of polycyclic aromatic hydrocarbon-DNA adducts in human tissues, has been modified to achieve approximately a 6-fold increase in sensitivity. The new assay, a competitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) has utilized the same rabbit antiserum as the ELISA, antiserum elicited against DNA modified with benzo[a]pyrene. However, the alkaline phosphatase conjugate has been replaced with a biotin-europium-labeled streptavidin signal amplification system, and the release of europium into the solution forms a highly fluorescent chelate complex that is measured by time-resolved fluorometry. The DELFIA has achieved a 5- to 6-fold increase in sensitivity for measurement of DNA samples modified in vitro with benzo[a]pyrene, for cultured cells exposed to radiolabeled benzo[a]pyrene, and for human samples from occupationally exposed workers. The assay has been validated by comparison of adduct levels determined by DELFIA, ELISA, and radioactivity in DNA from mouse keratinocytes exposed to radiolabeled benzo[a]pyrene. Human lymphocyte DNA samples from 104 Hungarian aluminum plant workers were assayed by ELISA and compared to blood cell DNA samples from 69 Italian coke oven workers assayed by DELFIA. The standard curves demonstrated that the limit of detection of 4.0 adducts in 10(8) nucleotides for polycyclic aromatic hydrocarbon-DNA adducts by ELISA, using 35 micrograms of DNA/microtiter plate well, has been decreased to 1.3 adducts in 10(8) nucleotides by DELFIA, using 20 micrograms of DNA/microtiter well. If 35 micrograms of DNA were used in the DELFIA, the calculated detection limit would be 0.7 adducts in 10(8) nucleotides.

Schoket, B.; Doty, W.A.; Vincze, I.; Strickland, P.T.; Ferri, G.M.; Assennato, G.; Poirier, M.C. (National Cancer Institute, NIH Bethesda, MD (United States))

1993-07-01

303

Molecular Species Identification with Rich Floristic Sampling: DNA Barcoding the Pteridophyte Flora of Japan  

PubMed Central

Background DNA barcoding is expected to be an effective identification tool for organisms with heteromorphic generations such as pteridophytes, which possess a morphologically simple gametophyte generation. Although a reference data set including complete coverage of the target local flora/fauna is necessary for accurate identification, DNA barcode studies including such rich taxonomic sampling on a countrywide scale are lacking. Methodology/Principal Findings The Japanese pteridophyte flora (733 taxa including subspecies and varieties) was used to test the utility of two plastid DNA barcode regions (rbcL and trnH-psbA) with the intention of developing an identification system for native gametophytes. DNA sequences were obtained from each of 689 (94.0%) taxa for rbcL and 617 (84.2%) taxa for trnH-psbA. Mean interspecific divergence values across all taxon pairs (K2P genetic distances) did not reveal a significant difference in rate between trnH-psbA and rbcL, but mean K2P distances of each genus showed significant heterogeneity according to systematic position. The minimum fail rate of taxon discrimination in an identification test using BLAST (12.52%) was obtained when rbcL and trnH-psbA were combined, and became lower in datasets excluding infraspecific taxa or apogamous taxa, or including sexual diploids only. Conclusions/Significance This study demonstrates the overall effectiveness of DNA barcodes for species identification in the Japanese pteridophyte flora. Although this flora is characterized by a high occurrence of apogamous taxa that pose a serious challenge to identification using DNA barcodes, such taxa are limited to a small number of genera, and only minimally detract from the overall success rate. In the case that a query sequence is matched to a known apogamous genus, routine species identification may not be possible. Otherwise, DNA barcoding is a practical tool for identification of most Japanese pteridophytes, and is especially anticipated to be helpful for identification of non-hybridizing gametophytes.

Ebihara, Atsushi; Nitta, Joel H.; Ito, Motomi

2010-01-01

304

Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil  

Microsoft Academic Search

Efficient, nonselective methods to obtain DNA from the environment are needed for rapid and thorough analysis of introduced microorganisms in environmental samples and for analysis of microbial community diversity in soil. A small-scale procedure to rapidly extract and purify DNA from soils was developed for in-the-field use. Amounts of DNA released from bacterial vegetative cells, bacterial endospores, and fungal conidia

CHERYL R. KUSKE; KAYSIE L. BANTON; DANTE L. ADORADA; PETER C. STARK; KAREN K. HILL; PAUL J. JACKSON

1998-01-01

305

Detection of the DNA of Borrelia afzelii, Anaplasma phagocytophilum and Babesia canis in blood samples from dogs in Warsaw.  

PubMed

Each month, from March 2003 to February 2004, 34 blood samples from dogs were randomly selected from the blood samples delivered to two veterinary laboratories in Warsaw and tested for the DNA of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Babesia canis and Hepatozoon canis. Borrelia DNA was detected in seven of the 408 dogs, A phagocytophilum DNA was found in two, and B canis DNA was found in 48 (11.8 per cent). The DNA of H canis was not found in any of the blood samples. Sequencing of the seven Borrelia amplicons showed that only the genospecies Borrelia afzelii was present, the first time it has been detected in dogs in Poland. PMID:19363228

Zygner, W; Górski, P; Wedrychowicz, H

2009-04-11

306

The TREX1 Exonuclease R114H Mutation in Aicardi-Gouti?res Syndrome and Lupus Reveals Dimeric Structure Requirements for DNA Degradation Activity*?  

PubMed Central

Mutations in the TREX1 gene cause Aicardi-Goutières syndrome (AGS) and are linked to the autoimmune disease systemic lupus erythematosus. The TREX1 protein is a dimeric 3? DNA exonuclease that degrades DNA to prevent inappropriate immune activation. One of the most common TREX1 mutations, R114H, causes AGS as a homozygous and compound heterozygous mutation and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1 proteins containing R114H and the insertion mutations aspartate at position 201 (D201ins) and alanine at position 124 (A124ins), found in compound heterozygous AGS with R114H, were prepared and the DNA degradation activities were tested. The homodimer TREX1R114H/R114H exhibits a 23-fold reduced single-stranded DNA (ssDNA) exonuclease activity relative to TREX1WT. The TREX1D201ins/D201ins and TREX1A124ins/A124ins exhibit more than 10,000-fold reduced ssDNA degradation activities. However, the TREX1R114H/D201ins and TREX1R114H/A124ins compound heterodimers exhibit activities 10-fold greater than the TREX1R114H/R114H homodimer during ssDNA and double-stranded DNA (dsDNA) degradation. These higher levels of activities measured in the TREX1R114H/D201ins and TREX1R114H/A124ins compound heterodimers are attributed to Arg-114 residues of TREX1D201ins and TREX1A124ins positioned at the dimer interface contributing to the active sites of the opposing TREX1R114H protomer. This interpretation is further supported by exonuclease activities measured for TREX1 enzymes containing R114A and R114K mutations. These biochemical data provide direct evidence for TREX1 residues in one protomer contributing to DNA degradation catalyzed in the opposing protomer and help to explain the dimeric TREX1 structure required for full catalytic competency.

Orebaugh, Clinton D.; Fye, Jason M.; Harvey, Scott; Hollis, Thomas; Perrino, Fred W.

2011-01-01

307

Targeted rapid amplification of cDNA ends (T-RACE)--an improved RACE reaction through degradation of non-target sequences  

PubMed Central

Amplification of the 5? ends of cDNA, although simple in theory, can often be difficult to achieve. We describe a novel method for the specific amplification of cDNA ends. An oligo-dT adapter incorporating a dUTP-containing PCR primer primes first-strand cDNA synthesis incorporating dUTP. Using the Cap finder approach, another distinct dUTP containing adapter is added to the 3? end of the newly synthesized cDNA. Second-strand synthesis incorporating dUTP is achieved by PCR, using dUTP-containing primers complimentary to the adapter sequences incorporated in the cDNA ends. The double-stranded cDNA-containing dUTP serves as a universal template for the specific amplification of the 3? or 5? end of any gene. To amplify the ends of cDNA, asymmetric PCR is performed using a single gene-specific primer and standard dNTPs. The asymmetric PCR product is purified and non-target transcripts containing dUTP degraded by Uracil DNA glycosylase, leaving only those transcripts produced during the asymmetric PCR. Subsequent PCR using a nested gene-specific primer and the 3? or 5? T-RACE primer results in specific amplification of cDNA ends. This method can be used to specifically amplify the 3? and 5? ends of numerous cDNAs from a single cDNA synthesis reaction.

Bower, Neil I.; Johnston, Ian A.

2010-01-01

308

Renewable Microcolumns for Automated DNA Purification and Flow-through Amplification: From Sediment Samples through Polymerase Chain Reaction  

SciTech Connect

There is an increasing need for field-portable systems for the detection and characterization of microorganisms in the environment. Nucleic acids analysis is frequently the method of choice for discriminating between bacteria in complex systems, but standard protocols are difficult to automate and current microfluidic devices are not configured specifically for environmental sample analysis. In this report, we describe the development of an integrated DNA purification and PCR amplification system and demonstrate its use for the automated purification and amplification of Geobacter chapelli DNA (genomic DNA or plasmid targets) from sediments. The system includes renewable separation columns for the automated capture and release of microparticle purification matrices, and can be easily reprogrammed for new separation chemistries and sample types. The DNA extraction efficiency for the automated system ranged from 3 to 25 percent, depending on the length and concentration of the DNA target . The system was more efficient than batch capture methods for the recovery of dilute genomic DNA even though the reagen volumes were smaller than required for the batch procedure. The automated DNA concentration and purification module was coupled to a flow-through, Peltier-controlled DNA amplification chamber, and used to successfully purify and amplify genomic and plasmid DNA from sediment extracts. Cleaning protocols were also developed to allow reuse of the integrated sample preparation system, including the flow-through PCR tube.

Bruckner-Lea, Cindy J. (BATTELLE (PACIFIC NW LAB)); Tsukuda, Toyoko (BATTELLE (PACIFIC NW LAB)); Dockendorff, Brian P. (BATTELLE (PACIFIC NW LAB)); Follansbee, James C. (BATTELLE (PACIFIC NW LAB)); Kingsley, Mark T. (BATTELLE (PACIFIC NW LAB)); Ocampo, Catherine O. (Pacific Northwest National Laboratory); Stults, Jennie R. (LOS ALAMOS TECH ASSOC); Chandler, Darrell P. (OFFICE OF FELLOWSHIP PROGRAMS)

2001-12-01

309

DNA degradation, UV sensitivity and SOS-mediated mutagenesis in strains of Escherichia coli deficient in single-strand DNA binding protein: Effects of mutations and treatments that alter levels of exonuclease V or RecA protein  

Microsoft Academic Search

Certain strains suppress the temperature-sensitivity caused by ssb-1, which encodes a mutant ssDNA binding protein (SSB). At 42°C, such strains are extremely UV-sensitive, degrade their DNA extensively after UV irradiation, and are deficient in UV mutability and UV induction of recA protein synthesis. We transduced recC22, which eliminates Exonuclease V activity, and recAo281, which causes operator-constitutive synthesis of recA protein,

Howard B. Lieberman; Evelyn M. Witkin

1983-01-01

310

SAMPLING DESIGN AND BIAS IN DNA-BASED CAPTURE–MARK–RECAPTURE POPULATION AND DENSITY ESTIMATES OF GRIZZLY BEARS  

Microsoft Academic Search

Over a 3-year period, we assessed 2 sampling designs for estimating grizzly bear (Ursus arctos) population size using DNA capture-mark-recapture methods on a population of bears that included radiomarked individuals. We compared a large-scale

JOHN BOULANGER; BRUCE N. MCLELLAN; JOHN G. WOODS; MICHAEL F. PROCTOR; CURTIS STROBECK; DeWoody

2004-01-01

311

Effects of sampling conditions on DNA-based estimates of American black bear abundance  

USGS Publications Warehouse

DNA-based capture-mark-recapture techniques are commonly used to estimate American black bear (Ursus americanus) population abundance (N). Although the technique is well established, many questions remain regarding study design. In particular, relationships among N, capture probability of heterogeneity mixtures A and B (pA and pB, respectively, or p, collectively), the proportion of each mixture (?), number of capture occasions (k), and probability of obtaining reliable estimates of N are not fully understood. We investigated these relationships using 1) an empirical dataset of DNA samples for which true N was unknown and 2) simulated datasets with known properties that represented a broader array of sampling conditions. For the empirical data analysis, we used the full closed population with heterogeneity data type in Program MARK to estimate N for a black bear population in Great Smoky Mountains National Park, Tennessee. We systematically reduced the number of those samples used in the analysis to evaluate the effect that changes in capture probabilities may have on parameter estimates. Model-averaged N for females and males were 161 (95% CI?=?114–272) and 100 (95% CI?=?74–167), respectively (pooled N?=?261, 95% CI?=?192–419), and the average weekly p was 0.09 for females and 0.12 for males. When we reduced the number of samples of the empirical data, support for heterogeneity models decreased. For the simulation analysis, we generated capture data with individual heterogeneity covering a range of sampling conditions commonly encountered in DNA-based capture-mark-recapture studies and examined the relationships between those conditions and accuracy (i.e., probability of obtaining an estimated N that is within 20% of true N), coverage (i.e., probability that 95% confidence interval includes true N), and precision (i.e., probability of obtaining a coefficient of variation ?20%) of estimates using logistic regression. The capture probability for the larger of 2 mixture proportions of the population (i.e., pA or pB, depending on the value of ?) was most important for predicting accuracy and precision, whereas capture probabilities of both mixture proportions (pA and pB) were important to explain variation in coverage. Based on sampling conditions similar to parameter estimates from the empirical dataset (pA?=?0.30, pB?=?0.05, N?=?250, ??=?0.15, and k?=?10), predicted accuracy and precision were low (60% and 53%, respectively), whereas coverage was high (94%). Increasing pB, the capture probability for the predominate but most difficult to capture proportion of the population, was most effective to improve accuracy under those conditions. However, manipulation of other parameters may be more effective under different conditions. In general, the probabilities of obtaining accurate and precise estimates were best when p??0.2. Our regression models can be used by managers to evaluate specific sampling scenarios and guide development of sampling frameworks or to assess reliability of DNA-based capture-mark-recapture studies.

Laufenberg, Jared S.; Van Manen, Frank T.; Clark, Joseph D.

2013-01-01

312

Initial clinical laboratory experience in noninvasive prenatal testing for fetal aneuploidy from maternal plasma DNA samples  

PubMed Central

Objective The aim of this study is to report the experience of noninvasive prenatal DNA testing using massively parallel sequencing in an accredited clinical laboratory. Methods Laboratory information was examined for blood samples received for testing between February and November 2012 for chromosome 21 (Chr21), Chr18, and Chr13. Monosomy X (MX) testing was available from July 2012 for cystic hygroma indication. Outcomes were collected from providers on samples with positive results. Results There were 5974 samples tested, and results were issued within an average of 5.1 business days. Aneuploidy was detected in 284 (4.8%) samples (155 Chr21, 66 Chr18, 19 Chr13, 40 MX, and four double aneuploidy). Follow-ups are available for 245/284 (86%), and 77/284 (27.1%) are confirmed, including one double-aneuploidy case concordant with cytogenetics from maternal malignancy. Fourteen (0.2%) discordant (putative false-positive) results (one Chr21, six Chr18, three Chr13, three MX, and one Chr21/13) have been identified. Five (0.08%) false-negative cases are reported (two trisomy 21, two trisomy 18, and one MX). In 170 (2.8%) cases, the result for a single chromosome was indefinite. Conclusions This report suggests that clinical testing of maternal cell-free DNA for fetal aneuploidy operates within performance parameters established in validation studies. Noninvasive prenatal testing is sensitive to biological contributions from placental and maternal sources. ©2013 Verinata Health, Inc. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Futch, Tracy; Spinosa, John; Bhatt, Sucheta; de Feo, Eileen; Rava, Richard P; Sehnert, Amy J

2013-01-01

313

Derivation of DNA probes for enumeration of a specific strain of Lactobacillus acidophilus in piglet digestive tract samples.  

PubMed Central

Four DNA probes were derived that hybridized specifically to DNA from Lactobacillus acidophilus O. The probes were constructed by randomly cloning lactobacillus DNA in plasmid vector pBR322. Two of the probes (pSR1 and pSR2) were composed of vector and plasmid DNA inserts (3.6 and 1.6 kb, respectively); the others (pSR3 and pSR4) were composed of vector and chromosomally derived inserts (6.9 and 1.4 kb, respectively). The probes were used to enumerate, by colony hybridization, strain O in digestive tract samples collected from piglets inoculated 24 hours previously with a culture of the strain. The probes did not hybridize to DNA from lactobacilli inhabiting the digestive tract of uninoculated piglets. Strain O made up about 10% of the total lactobacillus population of the pars esophagea and about 20% of the population in other digestive tract samples. Images

Rodtong, S; Dobbinson, S; Thode-Andersen, S; McConnell, M A; Tannock, G W

1993-01-01

314

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

PubMed

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 (AmpF?STR® 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. PMID:23254459

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

2013-07-01

315

Sampling  

NSDL National Science Digital Library

This tutorial covers some of the key terms in sampling like "population" and "sampling frame," some of the statistical terms used in sampling, and the major distinction between probability and Nonprobability sampling methods.

William Trochim (Cornell University)

2006-10-20

316

Expanding Character Sampling for Ciliate Phylogenetic Inference Using Mitochondrial SSU-rDNA as a Molecular Marker  

PubMed Central

Molecular systematics of ciliates, particularly at deep nodes, has largely focused on increasing taxon sampling using the nuclear small subunit rDNA (nSSU-rDNA) locus. These previous analyses have generally been congruent with morphologically-based classifications, although there is extensive non-monophyly at many levels. However, caution is needed in interpreting these results as nSSU-rDNA is just a single molecular marker. Here the mitochondrial small subunit rDNA (mtSSU-rDNA) is evaluated for deep ciliate nodes using the Colpodea as an example. Overall, well-supported nodes in the mtSSU-rDNA and concatenated topologies are well supported in the nSSU-rDNA topology; e.g., the non-monophyly of the Cyrtolophosidida. The two moderately-to well-supported incongruences between the loci are the placement of the Sorogenida and Colpoda aspera. Our analyses of mtSSU-rDNA support the conclusion, originally derived from nSSU-rDNA, that the morphological characters used in taxonomic circumscriptions of the Colpodea represent a mixture of ancestral and derived states. This demonstration of the efficacy of the mtSSU-rDNA will enable phylogenetic reconstructions of deep nodes in the ciliate tree of life to move from a single-locus to a multi-locus approach.

Dunthorn, Micah; Foissner, Wilhelm; Katz, Laura A.

2012-01-01

317

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

PubMed Central

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.

Schijman, Alejandro G.; Bisio, Margarita; Orellana, Liliana; Sued, Mariela; Duffy, Tomas; Mejia Jaramillo, Ana M.; Cura, Carolina; Auter, Frederic; Veron, Vincent; Qvarnstrom, Yvonne; Deborggraeve, Stijn; Hijar, Gisely; Zulantay, Ines; Lucero, Raul Horacio; Velazquez, Elsa; Tellez, Tatiana; Sanchez Leon, Zunilda; Galvao, Lucia; Nolder, Debbie; Monje Rumi, Maria; Levi, Jose E.; Ramirez, Juan D.; Zorrilla, Pilar; Flores, Maria; Jercic, Maria I.; Crisante, Gladys; Anez, Nestor; 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; Buscher, Philippe; Assal, Azzedine; Guhl, Felipe; Sosa Estani, Sergio; DaSilva, Alexandre; Britto, Constanca; Luquetti, Alejandro; Ladzins, Janis

2011-01-01

318

High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique  

SciTech Connect

Sample preparation has been one of the major bottlenecks for many high throughput analyses. The purpose of this research was to develop new sample preparation and integration approach for DNA sequencing, PCR based DNA analysis and combinatorial screening of homogeneous catalysis based on multiplexed capillary electrophoresis with laser induced fluorescence or imaging UV absorption detection. The author first introduced a method to integrate the front-end tasks to DNA capillary-array sequencers. protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. After the colony was picked, lysis was accomplished in situ in the plastic sample tube using either a thermocycler or heating block. Upon heating, the plasmids were released while chromsomal DNA and membrane proteins were denatured and precipitated to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed on-line DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation. For PCR based DNA analysis, they demonstrated that capillary electrophoresis with UV detection can be used for DNA analysis starting from clinical sample without purification. After PCR reaction using cheek cell, blood or HIV-1 gag DNA, the reaction mixtures was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis. The use of cheaper detection, and the elimination of purification of DNA sample before or after PCR reaction, will make this approach an attractive alternative to current methods for genetic analysis and disease diagnosis.

Yonghua Zhang

2002-05-27

319

Construction and Evaluation of Internal Control DNA for PCR Amplification of Chlamydia trachomatis DNA from Urine Samples  

Microsoft Academic Search

Received 25 July 2002\\/Returned for modification 12 October 2002\\/Accepted 12 December 2002 An internal control DNA (ICD) with the same primer binding sequences as the target Chlamydia trachomatis DNA was constructed and evaluated in a PCR assay with immunoenzymatic detection. One hundred urine specimens were tested, and 23 were found to contain inhibitors of the PCR, if not subjected to

Fotini Betsou; Katy Beaumont; Jean Marie Sueur; Jeanne Orfila

2003-01-01

320

A DNA based method to detect the grapevine root-rotting fungus Roesleria subterranea in soil and root samples  

PubMed Central

Summary Roesleria subterranea causes root rot in grapevine and fruit trees. The fungus has long been underestimated as a weak parasite, but during the last years it has been reported to cause severe damages in German vineyards. Direct, observation-based detection of the parasite is time consuming and destructive, as large parts of the rootstocks have to be uprooted and screened for the tiny, stipitate, hypogeous ascomata of R. subterranea. To facilitate rapid detection in vineyards, protocols to extract DNA from soil samples and grapevine roots, and R.-subterranea-specific PCR primers were designed. Twelve DNA–extraction protocols for soil samples were tested in small-scale experiments, and selected parameters were optimised. A protocol based on ball-mill homogenization, DNA extraction with SDS, skim milk, chloroform, and isopropanol, and subsequent purification of the raw extracts with PVPP-spin-columns was most effective. This DNA extraction protocol was found to be suitable for a wide range of soil-types including clay, loam and humic-rich soils. For DNA extraction from grapevine roots a CTAB-based protocol was more reliable for various grapevine rootstock varieties. Roesleria-subterranea-specific primers for the ITS1–5.8S–ITS2 rDNA-region were developed and tested for their specificity to DNA extracts from eleven R. subterranea strains isolated from grapevine and fruit trees. No cross reactions were detected with DNA extracts from 44 different species of fungi isolated from vineyard soils. The sensitivity of the species-specific primers in combination with the DNA extraction method for soil was high: as little as 100 fg ?l?1 R.-subterranea-DNA was sufficient for a detection in soil samples and plant material. Given that specific primers are available, the presented method will also allow quick and large-scale testing for other root pathogens.

Neuhauser, Sigrid; Huber, Lars; Kirchmair, Martin

2011-01-01

321

A DNA based method to detect the grapevine root-rotting fungus Roesleria subterranea in soil and root samples.  

PubMed

Roesleria subterranea causes root rot in grapevine and fruit trees. The fungus has long been underestimated as a weak parasite, but during the last years it has been reported to cause severe damages in German vineyards. Direct, observation-based detection of the parasite is time consuming and destructive, as large parts of the rootstocks have to be uprooted and screened for the tiny, stipitate, hypogeous ascomata of R. subterranea. To facilitate rapid detection in vineyards, protocols to extract DNA from soil samples and grapevine roots, and R.-subterranea-specific PCR primers were designed. Twelve DNA-extraction protocols for soil samples were tested in small-scale experiments, and selected parameters were optimised. A protocol based on ball-mill homogenization, DNA extraction with SDS, skim milk, chloroform, and isopropanol, and subsequent purification of the raw extracts with PVPP-spin-columns was most effective. This DNA extraction protocol was found to be suitable for a wide range of soil-types including clay, loam and humic-rich soils. For DNA extraction from grapevine roots a CTAB-based protocol was more reliable for various grapevine rootstock varieties. Roesleria-subterranea-specific primers for the ITS1-5.8S-ITS2 rDNA-region were developed and tested for their specificity to DNA extracts from eleven R. subterranea strains isolated from grapevine and fruit trees. No cross reactions were detected with DNA extracts from 44 different species of fungi isolated from vineyard soils. The sensitivity of the species-specific primers in combination with the DNA extraction method for soil was high: as little as 100 fg ?l(-1)R.-subterranea-DNA was sufficient for a detection in soil samples and plant material. Given that specific primers are available, the presented method will also allow quick and large-scale testing for other root pathogens. PMID:21442023

Neuhauser, Sigrid; Huber, Lars; Kirchmair, Martin

2009-08-01

322

Insights into biodiversity sampling strategies for freshwater microinvertebrate faunas through bioblitz campaigns and DNA barcoding  

PubMed Central

Background Biodiversity surveys have long depended on traditional methods of taxonomy to inform sampling protocols and to determine when a representative sample of a given species pool of interest has been obtained. Questions remain as to how to design appropriate sampling efforts to accurately estimate total biodiversity. Here we consider the biodiversity of freshwater ostracods (crustacean class Ostracoda) from the region of Churchill, Manitoba, Canada. Through an analysis of observed species richness and complementarity, accumulation curves, and richness estimators, we conduct an a posteriori analysis of five bioblitz-style collection strategies that differed in terms of total duration, number of sites, protocol flexibility to heterogeneous habitats, sorting of specimens for analysis, and primary purpose of collection. We used DNA barcoding to group specimens into molecular operational taxonomic units for comparison. Results Forty-eight provisional species were identified through genetic divergences, up from the 30 species previously known and documented in literature from the Churchill region. We found differential sampling efficiency among the five strategies, with liberal sorting of specimens for molecular analysis, protocol flexibility (and particularly a focus on covering diverse microhabitats), and a taxon-specific focus to collection having strong influences on garnering more accurate species richness estimates. Conclusions Our findings have implications for the successful design of future biodiversity surveys and citizen-science collection projects, which are becoming increasingly popular and have been shown to produce reliable results for a variety of taxa despite relying on largely untrained collectors. We propose that efficiency of biodiversity surveys can be increased by non-experts deliberately selecting diverse microhabitats; by conducting two rounds of molecular analysis, with the numbers of samples processed during round two informed by the singleton prevalence during round one; and by having sub-teams (even if all non-experts) focus on select taxa. Our study also provides new insights into subarctic diversity of freshwater Ostracoda and contributes to the broader “Barcoding Biotas” campaign at Churchill. Finally, we comment on the associated implications and future research directions for community ecology analyses and biodiversity surveys through DNA barcoding, which we show here to be an efficient technique enabling rapid biodiversity quantification in understudied taxa.

2013-01-01

323

Unexpected presence of Fagus orientalis complex in Italy as inferred from 45,000-year-old DNA pollen samples from Venice lagoon  

PubMed Central

Background Phylogeographic analyses on the Western Euroasiatic Fagus taxa (F. orientalis, F. sylvatica, F. taurica and F. moesiaca) is available, however, the subdivision of Fagus spp. is unresolved and there is no consensus on the phylogeny and on the identification (both with morphological than molecular markers) of Fagus Eurasiatic taxa. For the first time molecular analyses of ancient pollen, dated at least 45,000 years ago, were used in combination with the phylogeny analysis on current species, to identify the Fagus spp. present during the Last Interglacial period in Italy. In this work we aim at testing if the trnL-trnF chloroplast DNA (cpDNA) region, that has been previously proved efficient in discriminating different Quercus taxa, can be employed in distinguishing the Fagus species and in identifying the ancient pollen. Results 86 populations from 4 Western Euroasistic taxa were sampled, and sequenced for the trnL-trnF region to verify the efficiency of this cpDNA region in identifying the Fagus spp.. Furthermore, Fagus crenata (2 populations), Fagus grandifolia (2 populations), Fagus japonica, Fagus hayatae, Quercus species and Castanea species were analysed to better resolve the phylogenetic inference. Our results show that this cpDNA region harbour some informative sites that allow to infer relationships among the species within the Fagaceae family. In particular, few specific and fixed mutations were able to discriminate and identify all the different Fagus species. Considering a short fragment of 176 base pairs within the trnL intron, 2 transversions were found able in distinguishing the F. orientalis complex taxa (F. orientalis, F. taurica and F. moesiaca) from the remaining Fagus spp. (F. sylvatica, F. japonica, F. hayataea, F. crenata and F. grandifolia). This permits to analyse this fragment also in ancient samples, where DNA is usually highly degraded. The sequences data indicate that the DNA recovered from ancient pollen belongs to the F. orientalis complex since it displays the informative sites characteristic of this complex. Conclusion The ancient DNA sequences demonstrate for the first time that, in contrast to current knowledge based on palynological and macrofossil data, the F. orientalis complex was already present during the Tyrrhenian period in what is now the Venice lagoon (Italy). This is a new and important insight considering that nowadays West Europe is not the natural area of Fagus orientalis complex, and up to now nobody has hypothesized the presence during the Last Interglacial period of F. orientalis complex in Italy.

Paffetti, Donatella; Vettori, Cristina; Caramelli, David; Vernesi, Cristiano; Lari, Martina; Paganelli, Arturo; Paule, Ladislav; Giannini, Raffaello

2007-01-01

324

A novel methyl-binding domain protein enrichment method for identifying genome-wide tissue-specific DNA methylation from nanogram DNA samples  

PubMed Central

Background Growing evidence suggests that DNA methylation plays a role in tissue-specific differentiation. Current approaches to methylome analysis using enrichment with the methyl-binding domain protein (MBD) are restricted to large (?1 ?g) DNA samples, limiting the analysis of small tissue samples. Here we present a technique that enables characterization of genome-wide tissue-specific methylation patterns from nanogram quantities of DNA. Results We have developed a methodology utilizing MBD2b/MBD3L1 enrichment for methylated DNA, kinase pre-treated ligation-mediated PCR amplification (MeKL) and hybridization to the comprehensive high-throughput array for relative methylation (CHARM) customized tiling arrays, which we termed MeKL-chip. Kinase modification in combination with the addition of PEG has increased ligation-mediated PCR amplification over 20-fold, enabling >400-fold amplification of starting DNA. We have shown that MeKL-chip can be applied to as little as 20 ng of DNA, enabling comprehensive analysis of small DNA samples. Applying MeKL-chip to the mouse retina (a limited tissue source) and brain, 2,498 tissue-specific differentially methylated regions (T-DMRs) were characterized. The top five T-DMRs (Rgs20, Hes2, Nfic, Cckbr and Six3os1) were validated by pyrosequencing. Conclusions MeKL-chip enables genome-wide methylation analysis of nanogram quantities of DNA with a wide range of observed-to-expected CpG ratios due to the binding properties of the MBD2b/MBD3L1 protein complex. This methodology enabled the first analysis of genome-wide methylation in the mouse retina, characterizing novel T-DMRs.

2013-01-01

325

Amplification of Mitochondrial COII Gene from DNA Extracted from Hair Samples in Some Species of New World Monkeys  

Microsoft Academic Search

Nowadays, non‐invasive genotyping of wild and captive animals is the challenge [Morin et al., 1994; Constable et al., 2001]. Our group has been working for years on genetics of New World monkeys (NWM), employing blood samples as a source of DNA [Delprat et al., 1992; Ascunce et al., 2002], but we wanted to use alternative samples, such as hair and

Mariana S. Ascunce; Luciana Oklander; Marta D. Mudry

2003-01-01

326

DNA-PCR analysis of bloodstains sampled by the polyvinyl-alcohol method.  

PubMed

Among the usual techniques of sampling gunshot residues (GSR), the polyvinyl-alcohol method (PVAL) includes the advantage of embedding all particles, foreign bodies and stains on the surface of the shooter's hand in exact and reproducible topographic localization. The aim of the present study on ten persons killed by firearms was to check the possibility of DNA-PCR typing of blood traces embedded in the PVAL gloves in a second step following GSR analysis. The results of these examinations verify that the PVAL technique does not include factors that inhibit successful PCR typing. Thus the PVAL method can be recommended as a combination technique to secure and preserve inorganic and biological traces at the same time. PMID:9987876

Schyma, C; Huckenbeck, W; Bonte, W

1999-01-01

327

qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.

Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Gallati, Sabina, E-mail: sabina.gallati@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)] [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Schaller, Andre, E-mail: andre.schaller@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)] [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)

2012-07-06

328

Comparative Performance of Human Papillomavirus DNA Testing Using Novel Sample Collection Methods ?  

PubMed Central

To explore alternative cervical cancer screening approaches in an underserved population, we compared the performance of human papillomavirus (HPV) DNA assays in combination with different sample collection methods for primary cervical screening in the Mississippi Delta region. Three specimens were collected from women aged 26 to 65 years who were either routinely undergoing screening (n = 252) or not (n = 191): clinician-collected cervical specimens, clinician-collected cervicovaginal specimens, and self-collected cervicovaginal specimens taken at home. A novel collection device and medium were used for cervicovaginal sampling. Specimens were tested by three HPV DNA assays: hybrid capture 2 (HC2; Qiagen Corp., Gaithersburg, MD), Linear Array (LA; Roche Molecular Systems, Pleasanton, CA), and Amplicor (Roche Molecular Systems, Pleasanton, CA). Liquid-based cytology was performed on cervical specimens. We compared the overall positivity (a proxy for clinical specificity) for any carcinogenic HPV genotype and calculated the agreement across assay and specimen type using McNemar's test for differences in test positivity. Across all three assays there were no significant differences between clinician-collected and self-collected cervicovaginal specimens (P > 0.01 for all comparisons). For both cervicovaginal specimens (clinician collected and self-collected), fewer women tested positive by HC2 than by LA or Amplicor (P < 0.01 for all comparisons). HC2 had the best agreement between specimens for all assays. HC2 is likely more clinically specific, although possibly less sensitive, than either PCR test. Thus, use of HC2 on cervicovaginal specimens for screening could result in fewer referrals compared to LA and Amplicor.

Gage, Julia C.; Partridge, Edward E.; Rausa, Alfio; Gravitt, Patti E.; Wacholder, Sholom; Schiffman, Mark; Scarinci, Isabel; Castle, Philip E.

2011-01-01

329

Tagging the Signatures of Domestication in Common Bean (Phaseolus vulgaris) by Means of Pooled DNA Samples  

PubMed Central

Background and Aims The main aim of this study was to use an amplified fragment length polymorphism (AFLP)-based, large-scale screening of the whole genome of Phaseolus vulgaris to determine the effects of selection on the structure of the genetic diversity in wild and domesticated populations. Methods Using pooled DNA samples, seven each of wild and domesticated populations of P. vulgaris were studied using 2506 AFLP markers (on average, one every 250 kb). About 10 % of the markers were also analysed on individual genotypes and were used to infer allelic frequencies empirically from bulk data. In both data sets, tests were made to determine the departure from neutral expectation for each marker using an FST-based method. Key Results The most important outcome is that a large fraction of the genome of the common bean (16 %; P < 0·01) appears to have been subjected to effects of selection during domestication. Markers obtained in individual genotypes were also mapped and classified according to their proximities to known genes and quantitative trait loci (QTLs) of the domestication syndrome. Most of the markers that were found to be potentially under the effects of selection were located in the proximity of previously mapped genes and QTLs related to the domestication syndrome. Conclusions Overall, the results indicate that in P. vulgaris a large portion of the genome appears to have been subjected to the effects of selection, probably because of linkage to the loci selected during domestication. As most of the markers that are under the effects of selection are linked to known loci related to the domestication syndrome, it is concluded that population genomics approaches are very efficient in detecting QTLs. A method based on bulk DNA samples is presented that is effective in pre-screening for a large number of markers to determine selection signatures.

Papa, Roberto; Bellucci, Elisa; Rossi, Monica; Leonardi, Stefano; Rau, Domenico; Gepts, Paul; Nanni, Laura; Attene, Giovanna

2007-01-01

330

Use of various Fe-modified montmorillonite samples for 4-nitrophenol degradation by H 2O 2  

Microsoft Academic Search

4-nitrophenol is degraded by H2O2 with iron (III)-exchanged and pillared montmorillonite as catalysts. The influence of different types of catalysts, their amounts and the H2O2 concentration were investigated.At optimal conditions, a solution containing 10?3 M of 4-nitrophenol and 10?2 M of H2O2 was degraded in less than 6 h in the presence of (1 g\\/l) of mixed (Al–Fe) pillared montmorillonite

L Chirchi; A Ghorbel

2002-01-01

331

A novel method of selective removal of human DNA improves PCR sensitivity for detection of Salmonella Typhi in blood samples  

PubMed Central

Background Enteric fever is a major public health problem, causing an estimated 21million new cases and 216,000 or more deaths every year. Current diagnosis of the disease is inadequate. Blood culture only identifies 45 to 70% of the cases and is time-consuming. Serological tests have very low sensitivity and specificity. Clinical samples obtained for diagnosis of enteric fever in the field generally have <1 organism/ml of blood, so that even PCR-based methods, widely used for detection of other infectious diseases, are not a straightforward option in typhoid diagnosis. We developed a novel method to enrich target bacterial DNA by selective removal of human DNA from blood samples, enhancing the sensitivity of PCR tests. This method offers the possibility of improving PCR assays directly using clinical specimens for diagnosis of this globally important infectious disease. Methods Blood samples were mixed with ox bile for selective lysis of human blood cells and the released human DNA was then digested with addition of bile resistant micrococcal nuclease. The intact Salmonella Typhi bacteria were collected from the specimen by centrifugation and the DNA extracted with QIAamp DNA mini kit. The presence of Salmonella Typhi bacteria in blood samples was detected by PCR with the fliC-d gene of Salmonella Typhi as the target. Results Micrococcal nuclease retained activity against human blood DNA in the presence of up to 9% ox bile. Background human DNA was dramatically removed from blood samples through the use of ox bile lysis and micrococcal nuclease for removal of mammalian DNA. Consequently target Salmonella Typhi DNA was enriched in DNA preparations and the PCR sensitivity for detection of Salmonella Typhi in spiked blood samples was enhanced by 1,000 fold. Conclusions Use of a combination of selective ox-bile blood cell lysis and removal of human DNA with micrococcal nuclease significantly improves PCR sensitivity and offers a better option for improved typhoid PCR assays directly using clinical specimens in diagnosis of this globally important infection disease which we believe could be of importance in improving clinical care and providing effective evaluation of novel vaccines.

2012-01-01

332

A comparison study between a disposable electrochemical DNA biosensor and a Vibrio fischeri-based luminescent sensor for the detection of toxicants in water samples  

Microsoft Academic Search

In the present study, a comparison between a disposable electrochemical DNA biosensor and a Vibrio fischeri-based luminescent sensor for the detection of toxicants in water samples was made.In order to realize this study, a disposable electrochemical DNA biosensor has been reported. The DNA biosensor is assembled by immobilizing double stranded Calf Thymus DNA onto the surface of a disposable carbon

Anca Monica Tencaliec; Serena Laschi; Vasile Magearu; Marco Mascini

2006-01-01

333

Application of group specific amplified rDNA restriction analysis to characterize swine fecal and manure storage pit samples.  

PubMed

Group specific amplified ribosomal-DNA restriction analysis was evaluated as a method to rapidly assess microbial community structure in swine fecal and manure storage pit samples. PCR primer sequences were evaluated for their specificity to ribosomal DNA from selected bacterial groups by optimizing annealing temperatures and determining specificity using a set of primer target and non-target organisms. A number of primer sets were identified targeting the following groups: Bacteroides-Prevotella, clostridial clusters I and II, clostridial clusters IX and XI, clostridial clusters XIVa and XIVb, Lactobacillus, Desulfovibrionaceae and Streptococcus-Lactococcus, as well as an universal primer set to represent total populations. Each bacterial group was digested with at least three restriction enzymes. We applied the group specific amplified ribosomal-DNA restriction analysis to swine fecal and manure storage pit samples obtained on two separate occasions. Fecal and manure storage pit samples obtained on the same day were more similar to each other than to any other samples. Results were consistent with 16S ribosomal DNA sequencing data from bacterial isolates and clones obtained from swine feces and manure storage pit. The group specific amplified ribosomal-DNA restriction analysis technique was able to rapid detect gross bacterial community differences among swine fecal and manure storage pit samples and determine groups of interest for more detailed examination. PMID:16701521

Ziemer, C J; Cotta, M A; Whitehead, T R

2004-08-01

334

Inferring separate parental admixture components in unknown DNA samples using autosomal SNPs.  

PubMed

The identification of ancestral admixture proportions for human DNA samples has recently had success in forensic cases. Current methods infer admixture proportions for the target sample, but not for their parents, which provides an additional layer of information that may aid certain forensic investigations. We describe new maximum likelihood methods (LEAPFrOG and LEAPFrOG Expectation Maximisation), for inferring both an individual's admixture proportions and the admixture proportions possessed by the unobserved parents, with respect to two or more source populations, using single-nucleotide polymorphism data typed only in the target individual. This is achieved by examining the increase in heterozygosity in the offspring of parents who are from different populations or who represent different mixtures from a number of source populations. We validated the methods via simulation; combining chromosomes from different Hapmap Phase III population samples to emulate first-generation admixture. Performance was strong for individuals with mixed African/European (YRI/CEU) ancestry, but poor for mixed Japanese/Chinese (JPT/CHB) ancestry, reflecting the difficulty in distinguishing closely related source populations. A total of 11 African-American trios were used to compare the parental admixture inferred from their own genotypes against that inferred purely from their offspring genotypes. We examined the performance of 34 ancestry informative markers from a multiplex kit for ancestry inference. Simulations showed that estimates were unreliable when parents had similar admixture, suggesting more markers are needed. Our results demonstrate that ancestral backgrounds of case samples and their parents are obtainable to aid in forensic investigations, provided that high-throughput methods are adopted by the forensic community. PMID:22739346

Crouch, Daniel J M; Weale, Michael E

2012-12-01

335

Identification of Eukaryotic Open Reading Frames in Metagenomic cDNA Libraries Made from Environmental Samples  

PubMed Central

Here we describe the application of metagenomic technologies to construct cDNA libraries from RNA isolated from environmental samples. RNAlater (Ambion) was shown to stabilize RNA in environmental samples for periods of at least 3 months at ?20°C. Protocols for library construction were established on total RNA extracted from Acanthamoeba polyphaga trophozoites. The methodology was then used on algal mats from geothermal hot springs in Tengchong county, Yunnan Province, People's Republic of China, and activated sludge from a sewage treatment plant in Leicestershire, United Kingdom. The Tenchong libraries were dominated by RNA from prokaryotes, reflecting the mainly prokaryote microbial composition. The majority of these clones resulted from rRNA; only a few appeared to be derived from mRNA. In contrast, many clones from the activated sludge library had significant similarity to eukaryote mRNA-encoded protein sequences. A library was also made using polyadenylated RNA isolated from total RNA from activated sludge; many more clones in this library were related to eukaryotic mRNA sequences and proteins. Open reading frames (ORFs) up to 378 amino acids in size could be identified. Some resembled known proteins over their full length, e.g., 36% match to cystatin, 49% match to ribosomal protein L32, 63% match to ribosomal protein S16, 70% to CPC2 protein. The methodology described here permits the polyadenylated transcriptome to be isolated from environmental samples with no knowledge of the identity of the microorganisms in the sample or the necessity to culture them. It has many uses, including the identification of novel eukaryotic ORFs encoding proteins and enzymes.

Grant, Susan; Grant, William D.; Cowan, Don A.; Jones, Brian E.; Ma, Yanhe; Ventosa, Antonio; Heaphy, Shaun

2006-01-01

336

Functions of the DNA damage response pathway target Ho endonuclease of yeast for degradation via the ubiquitin-26S proteasome system  

Microsoft Academic Search

Ho endonuclease of Saccharomyces cerevisiae is a homing endonuclease that makes a site-specific double-strand break in the MAT gene in late G1. Here we show that Ho is rapidly degraded via the ubiquitin-26S proteasome system through two ubiquitin-conjugating enzymes UBC2Rad6 and UBC3Cdc34. UBC2Rad6 is complexed with the ring finger DNA-binding protein Rad18, and we find that Ho is stabilized in

Ludmila Kaplun; Yelena Ivantsiv; Daniel Kornitzer; Dina Raveh

2000-01-01

337

Def1 promotes the degradation of Pol3 for polymerase exchange to occur during DNA-damage--induced mutagenesis in Saccharomyces cerevisiae.  

PubMed

DNA damages hinder the advance of replication forks because of the inability of the replicative polymerases to synthesize across most DNA lesions. Because stalled replication forks are prone to undergo DNA breakage and recombination that can lead to chromosomal rearrangements and cell death, cells possess different mechanisms to ensure the continuity of replication on damaged templates. Specialized, translesion synthesis (TLS) polymerases can take over synthesis at DNA damage sites. TLS polymerases synthesize DNA with a high error rate and are responsible for damage-induced mutagenesis, so their activity must be strictly regulated. However, the mechanism that allows their replacement of the replicative polymerase is unknown. Here, using protein complex purification and yeast genetic tools, we identify Def1 as a key factor for damage-induced mutagenesis in yeast. In in vivo experiments we demonstrate that upon DNA damage, Def1 promotes the ubiquitylation and subsequent proteasomal degradation of Pol3, the catalytic subunit of the replicative polymerase ?, whereas Pol31 and Pol32, the other two subunits of polymerase ?, are not affected. We also show that purified Pol31 and Pol32 can form a complex with the TLS polymerase Rev1. Our results imply that TLS polymerases carry out DNA lesion bypass only after the Def1-assisted removal of Pol3 from the stalled replication fork. PMID:24465179

Daraba, Andreea; Gali, Vamsi K; Halmai, Miklós; Haracska, Lajos; Unk, Ildiko

2014-01-01

338

Development of tetranucleotide microsatellite loci and a non-invasive DNA sampling method for Texas horned lizards ( Phrynosoma cornutum )  

Microsoft Academic Search

We developed a non-invasive DNA sampling method and 15 tetranucleotide microsatellite markers for Texas horned lizards (Phrynosoma cornutum). Swabbing the cloaca with a small cotton swab and preserving the cells in lysis buffer was an effective method to obtain\\u000a tissue for DNA extraction. Loci were highly polymorphic with 8–25 alleles and observed heterozygosity was high (0.71–0.96).\\u000a Some of these loci

Dean A. Williams; Cory Leach; Amanda M. Hale; Kristopher B. Karsten; Emmanuela Mujica; Diane Barber; Lee Ann Linam; Nathan Rains

339

Routine acid decalcification of bone marrow samples can preserve DNA for FISH and CGH studies in metastatic prostate cancer.  

PubMed

Production of paraffin-section material from tissue samples that contain bone requires decalcification. Techniques such as acidic decalcification or EDTA chelation are suitable methods. Acid decalcification is generally quicker than EDTA chelation but studies have suggested that it may result in hydrolysis of DNA. Here we show that limited acid decalcification (less than 24 hr) in 5% formic acid can preserve DNA sufficient for fluorescent in situ hybridization (FISH) or comparative genomic hybridization (CGH) and that prolonged 10% formic acid decalcification results in failure of FISH and only limited retrieval of DNA for CGH studies. PMID:11748301

Brown, R S D; Edwards, J; Bartlett, J W; Jones, C; Dogan, A

2002-01-01

340

Mitochondrial DNA Damage and Dysfunction, and Oxidative Stress Are Associated with Endoplasmic Reticulum Stress, Protein Degradation and Apoptosis in High Fat Diet-Induced Insulin Resistance Mice  

PubMed Central

Background Recent studies showed a link between a high fat diet (HFD)-induced obesity and lipid accumulation in non-adipose tissues, such as skeletal muscle and liver, and insulin resistance (IR). Although the mechanisms responsible for IR in those tissues are different, oxidative stress and mitochondrial dysfunction have been implicated in the disease process. We tested the hypothesis that HFD induced mitochondrial DNA (mtDNA) damage and that this damage is associated with mitochondrial dysfunction, oxidative stress, and induction of markers of endoplasmic reticulum (ER) stress, protein degradation and apoptosis in skeletal muscle and liver in a mouse model of obesity-induced IR. Methodology/Principal Findings C57BL/6J male mice were fed either a HFD (60% fat) or normal chow (NC) (10% fat) for 16 weeks. We found that HFD-induced IR correlated with increased mtDNA damage, mitochondrial dysfunction and markers of oxidative stress in skeletal muscle and liver. Also, a HFD causes a change in the expression level of DNA repair enzymes in both nuclei and mitochondria in skeletal muscle and liver. Furthermore, a HFD leads to activation of ER stress, protein degradation and apoptosis in skeletal muscle and liver, and significantly reduced the content of two major proteins involved in insulin signaling, Akt and IRS-1 in skeletal muscle, and Akt in liver. Basal p-Akt level was not significantly influenced by HFD feeding in skeletal muscle and liver. Conclusions/Significance This study provides new evidence that HFD-induced mtDNA damage correlates with mitochondrial dysfunction and increased oxidative stress in skeletal muscle and liver, which is associated with the induction of markers of ER stress, protein degradation and apoptosis.

Yuzefovych, Larysa V.; Musiyenko, Sergiy I.; Wilson, Glenn L.; Rachek, Lyudmila I.

2013-01-01

341

A one step multiplex PCR assay for rapid screening of East Asian mtDNA haplogroups on forensic samples.  

PubMed

The mitochondrial DNA (mtDNA) haplogroup typing has become an essential tool to study human evolutionary history and to infer the matrilineal bio-geographic ancestry. In forensic field, the screening of mtDNA haplogroups by genotyping of mtDNA single nucleotide polymorphisms (SNPs) can help guarantee the quality of mtDNA sequence data as well as can reduce the need to sequence samples that do not match. Here, a multiplex mutagenically separated (MS) polymerase chain reaction (PCR) system was developed for simultaneous rapid detection of 14 coding region SNPs and one deletion motif representing common mtDNA haplogroups of East Asia. The multiplex MS PCR system we developed has the advantage of being a one step procedure that requires only a single PCR amplification with allele-specific primers and allowing straightforward designation of haplogroups along the branches of the phylogenetic tree. Therefore, it would be a simple, rapid, and reliable detection method useful for large-scale screening of mtDNA variations to determine East Asian mtDNA haplogroups. PMID:22981178

Lee, Hwan Young; Yoon, Jung Ah; Yang, Woo Ick; Shin, Kyoung-Jin

2013-01-01

342

Development of a quality, high throughput DNA analysis procedure for skeletal samples to assist with the identification of victims from the World Trade Center attacks.  

PubMed

The attacks on the World Trade Center (WTC) Towers on September 11, 2001, represented the single largest terrorist-related mass fatality incident in the history of the United States. More than 2,700 individuals of varied racial and ethnic background lost their lives that day. Through the efforts of thousands of citizens, including recovery workers, medical examiners, and forensic scientists, the identification of approximately 1,500 victims had been accomplished through June 2003 (the majority of these identifications were made within the first 8-12 months). The principal role of The Bode Technology Group (Bode) in this process was to develop a quality, high throughput DNA extraction and short tandem repeat (STR) analysis procedure for skeletal elements, and to provide STR profiles to the Office of the Chief Medical Examiner (OCME) in New York City to be used for identification of the victims. A high throughput process was developed to include electronic accessioning of samples, so that the numbering system of the OCME was maintained; rapid preparation and sampling of skeletal fragments to allow for the processing of more than 250 fragments per day; use of a 96-well format for sample extraction, DNA quantification, and STR analysis; and use of the Applied Biosystems 3100 and 3700 instrumentation to develop STR profiles. Given the highly degraded nature of the skeletal remains received by Bode, an advanced DNA extraction procedure was developed to increase the quantity of DNA recovery and reduce the co-purification of polymerase chain reaction (PCR) amplification inhibitors. In addition, two new STR multiplexes were developed specifically for this project, which reduced the amplicon size of the STR loci, and therefore, enhanced the ability to obtain results from the most challenged of samples. In all, the procedures developed allowed for the analysis of more than 1,000 skeletal samples each week. Approximately 13,000 skeletal fragments were analyzed at least once, for a total of more than 18,000 analyses, and greater than 8,000 of the skeletal samples produced STR results (65%). The percentage of successful results was low in relation to previous mass fatality incidents involving airline disasters. However, when this same process was applied to the analysis of skeletal remains from the American Airlines Flight 587 disaster that occurred on November 12, 2001, the success rate was in line with expected results (ie, greater than 92% of the skeletal remains produced results). This illustrated the quality aspects of the procedure and the degree of degradation that had occurred for the remains of the WTC victims. For future mass fatality incidents, the quality, high throughput procedures developed by Bode will allow for more rapid DNA analysis of victim remains, more rapid identification of victims, and thus more rapid return of remains to family members. PMID:12808717

Holland, Mitchell M; Cave, Christopher A; Holland, Charity A; Bille, Todd W

2003-06-01

343

New type of SSUrDNA sequence was detected from both Plasmodium ovale curtisi and Plasmodium ovale wallikeri samples  

PubMed Central

Background Plasmodium ovale is relatively unfamiliar to Chinese staff engaged in malaria diagnosis. In 2013, dried blood spots of four unidentified but suspected ovale malaria samples were sent to the National Malaria Reference Laboratory (NMRL) for reconfirmation. Methods Partial and complete, small, subunit ribosomal DNA (SSU rDNA) sequences of four samples were obtained with PCR-cloning-sequencing method. Obtained sequences were analyzed by aligning with each other and with nine SSU rDNA sequences of six known Plasmodium parasites. A phylogenetic tree was constructed based on complete SSU rDNA sequences and 12 same gene sequences derived from six known Plasmodium parasites and three Babesia parasites. Primary structure of conservative and variable regions of variant sequences was determined also by comparing them with those of six known Plasmodium parasites. To confirm their existence in genome, they were redetected with primers matching their variable regions. PCR systems aimed to roughly detect any eukaryotes and prokaryotes respectively were also applied to search for other pathogens in one of four patients. Results Totally, 19 partial and 23 complete SSU rDNA sequences obtained from four samples. Except eight variant sequences, similarities among sequences from same DNA sample were in general high (more than 98%). The phylogenetic analysis revealed that three cases were infected by P. ovale wallikeri and one by P. ovale curtisi. Four of the variant sequences which obtained from four samples relatively showed high similarities with each other (98.5%-100%). Identical variant sequences actually could be re-obtained from each DNA sample. Their primary structure of conservative and variable regions showed quite fit with that of six known Plasmodium parasites. The test for prokaryote pathogens showed negative and the tests for eukaryotes only found DNA sequences of Human and P. ovale parasites. Conclusion Both P. ovale wallikeri and P. ovale curtisi infections are present in imported malaria cases of China. New type of partial SSU rDNA sequence which assumed to express in a certain life stage of P. ovale was obtained from both P. ovale wallikeri and P. ovale curtisi samples. This discovery would supply information and clues to identify and understand P. ovale parasites more accurately.

2014-01-01

344

Spectral, thermal, kinetic, molecular modeling and eukaryotic DNA degradation studies for a new series of albendazole (HABZ) complexes.  

PubMed

This work represents the elaborated investigation for the ligational behavior of the albendazole ligand through its coordination with, Cu(II), Mn(II), Ni(II), Co(II) and Cr(III) ions. Elemental analysis, molar conductance, magnetic moment, spectral studies (IR, UV-Vis and ESR) and thermogravimetric analysis (TG and DTG) have been used to characterize the isolated complexes. A deliberate comparison for the IR spectra reveals that the ligand coordinated with all mentioned metal ions by the same manner as a neutral bidentate through carbonyl of ester moiety and NH groups. The proposed chelation form for such complexes is expected through out the preparation conditions in a relatively acidic medium. The powder XRD study reflects the amorphous nature for the investigated complexes except Mn(II). The conductivity measurements reflect the non-electrolytic feature for all complexes. In comparing with the constants for the magnetic measurements as well as the electronic spectral data, the octahedral structure was proposed strongly for Cr(III) and Ni(II), the tetrahedral for Co(II) and Mn(II) complexes but the square-pyramidal for the Cu(II) one. The thermogravimetric analysis confirms the presence or absence of water molecules by any type of attachments. Also, the kinetic parameters are estimated from DTG and TG curves. ESR spectrum data for Cu(II) solid complex confirms the square-pyramidal state is the most fitted one for the coordinated structure. The albendazole ligand and its complexes are biologically investigated against two bacteria as well as their effective effect on degradation of calf thymus DNA. PMID:20934909

El-Metwaly, Nashwa M; Refat, Moamen S

2011-01-01

345

Synthesis, characterization, molecular modeling and eukaryotic DNA degradation of 1-(3,4-dihydroxybenzylidene)thiosemicarbazide complexes  

NASA Astrophysics Data System (ADS)

A new chelating agent, 1-(3,4-dihydroxybenzylidene)thiosemicarbazide, H 3BTS, has been introduced for complexation with AsO 2+, SbO +, VO 2+, Cr(III), Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Bi(III) ions. The isolated chelates are characterized by partial elemental analyses, magnetic moments, spectra (IR, UV-Vis, ESR) and thermal studies. The molecular parameters of the ligand and its metal complexes have been calculated. The protonation constants of H 3BTS (10.35, 9.45 and 8.35) and the stepwise stability constants of its complexes are calculated; Fe(III)-H 3BTS system was found the most stable while Cr(III)-H 3BTS was the lowest. The ligand coordinates as tribasic with Fe(III), Ni(II) and SbO +, dibasic with VO 2+, Cu(II), Co(II), Bi(III), AsO 2+ and Cr(III) and monobasic with Zn(II) ions. Copper(II) and chromium(III) complexes measured anomalous magnetic moments while Co(II), Ni(II) and Fe(III) complexes have normal values. The ligand field parameters were calculated for [Cr 2(HL)(OAc) 2(OH) 2(H 2O)] and [Co(HL)(H 2O) 2] and their values were found in the range reported for a tetrahedral structure. The ESR spectra of the Cu(II) and VO 2+ complexes support the binuclear structure. The end product in the thermal decomposition of some complexes are the metals (Bi, As and/or Sb). [(VO) 2(HL)(SO 4)]2H 2O, [Cr 2(HL)(OAc) 2(OH) 2(H 2O)] and [Co(HL)(H 2O) 2] are found active for the degradation of the DNA of eukaryotic subject completely.

El-Asmy, Ahmed A.; Al-Gammal, Ola A.; Saad, Dena A.; Ghazy, Shaban E.

2009-09-01

346

Spectral, thermal, kinetic, molecular modeling and eukaryotic DNA degradation studies for a new series of albendazole (HABZ) complexes  

NASA Astrophysics Data System (ADS)

This work represents the elaborated investigation for the ligational behavior of the albendazole ligand through its coordination with, Cu(II), Mn(II), Ni(II), Co(II) and Cr(III) ions. Elemental analysis, molar conductance, magnetic moment, spectral studies (IR, UV-Vis and ESR) and thermogravimetric analysis (TG and DTG) have been used to characterize the isolated complexes. A deliberate comparison for the IR spectra reveals that the ligand coordinated with all mentioned metal ions by the same manner as a neutral bidentate through carbonyl of ester moiety and NH groups. The proposed chelation form for such complexes is expected through out the preparation conditions in a relatively acidic medium. The powder XRD study reflects the amorphous nature for the investigated complexes except Mn(II). The conductivity measurements reflect the non-electrolytic feature for all complexes. In comparing with the constants for the magnetic measurements as well as the electronic spectral data, the octahedral structure was proposed strongly for Cr(III) and Ni(II), the tetrahedral for Co(II) and Mn(II) complexes but the square-pyramidal for the Cu(II) one. The thermogravimetric analysis confirms the presence or absence of water molecules by any type of attachments. Also, the kinetic parameters are estimated from DTG and TG curves. ESR spectrum data for Cu(II) solid complex confirms the square-pyramidal state is the most fitted one for the coordinated structure. The albendazole ligand and its complexes are biologically investigated against two bacteria as well as their effective effect on degradation of calf thymus DNA.

El-Metwaly, Nashwa M.; Refat, Moamen S.

2011-01-01

347

Majority of divergence between closely related DNA samples is due to indels  

Microsoft Academic Search

utations in the DNA are the source of variation in Darwinian evolution. Therefore it is likely that the exam- ination of DNA differences between closely related species or among polymorphic variations in DNA of a given species will give insight into the nature of the mutations and the process of evolution. In the present paper, published and unpublished data are

Roy J. Britten; Lee Rowen; John Williams; R. Andrew Cameron

2003-01-01

348

What technique should be used for routine detection and quantification of HBV DNA in clinical samples?  

Microsoft Academic Search

Detection of hepatitis B virus (HBV) DNA in serum allows monitoring of HBV replication and assessing responses to antiviral treatment. HBV DNA quantification measures virus replication and can be used as a prognosis indicator of liver disease and an index of response to antiviral drugs. The aim of this study was to compare the performances of three HBV DNA detection

Jean-Michel Pawlotsky; Anne Bastie; Isabelle Lonjon; Jocelyne Re´mire´; Françoise Darthuy; Claude-James Soussy; Daniel Dhumeaux

1997-01-01

349

Rapid Diagnosis of Extrapulmonary Tuberculosis by PCR: Impact of Sample Preparation and DNA Extraction  

PubMed Central

In cases of suspected extrapulmonary tuberculosis, rapid and accurate laboratory diagnosis is of prime importance, since traditional techniques of detecting acid-fast bacilli have limitations. The major difficulty with mycobacteria is achieving optimal cell lysis. Buffers used in commercial kits do not allow this complete lysis in a number of clinical specimens. A comparison of two sample preparation methods, pretreatment with proteinase K (PK-Roche) and complete DNA purification (cetyltrimethylammonium bromide [CTAB]-Roche), was conducted on 144 extrapulmonary specimens collected from 120 patients to evaluate the impact on the Cobas-Amplicor method. Thirty patients were diagnosed with tuberculosis, with 15 patients culture positive for Mycobacterium tuberculosis. Amplification and detection of the amplicons were impaired by a high number of inhibitory specimens (39 to 52%). CTAB-Roche allowed the detection of more culture-positive specimens by PCR than PK-Roche. Comparison with the final diagnoses of tuberculosis confirmed that CTAB-Roche produced the best sensitivity (53.8%) compared to culture (43.3%), PK-Roche (16%), and smear (13%). However, the specificity of the PCR assay with CTAB-Roche-extracted material was always lower (78.8%) than those with culture (100%) and PK-Roche (96.5%). False-positive specimens were lung biopsy material, lymph node biopsy material and aspirate, or bone marrow aspirate, mainly from immunocompromised patients. Despite the efficiency of complete DNA extraction for the rapid diagnosis by PCR of extrapulmonary tuberculosis, the false-positive results challenge our understanding of PCR results.

Honore-Bouakline, S.; Vincensini, J. P.; Giacuzzo, V.; Lagrange, P. H.; Herrmann, J. L.

2003-01-01

350

Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing  

PubMed Central

Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy.

Be, Nicholas A.; Thissen, James B.; Gardner, Shea N.; McLoughlin, Kevin S.; Fofanov, Viacheslav Y.; Koshinsky, Heather; Ellingson, Sally R.; Brettin, Thomas S.; Jackson, Paul J.; Jaing, Crystal J.

2013-01-01

351

An investigation into the performance of methods for adjusting for sampling uncertainty in DNA likelihood ratio calculations.  

PubMed

There is a variety of methods for assessing sampling uncertainty in likelihood ratio calculations in DNA casework. Sampling uncertainty arises because all DNA statistical methods rely on a database of collected profiles. Such databases can be regarded as a sample from the population of interest. The act of taking a sample incurs sampling uncertainty. In some circumstances it may be desirable to provide some estimate of this uncertainty. We have addressed this topic in two previous publications [1,2]. In this paper we reconsider the performance of the methods using 15 locus Identifiler™ profiles, rather than the 6 locus data used in [1]. We also examine the differences in performance observed when using a uniform prior versus a 1/k prior in the Bayesian highest posterior density (HPD) method of Curran et al. [1]. PMID:21208838

Curran, James M; Buckleton, John S

2011-11-01

352

New procedure for recovering extra- and intracellular DNA from marine sediment samples  

NASA Astrophysics Data System (ADS)

Extracellular DNA (eDNA) is a ubiquitous biological compound in aquatic sediment and soil. Despite major methodological advances, analysis of DNA from sediment is still technically challenging, not just because of the co-elution of inhibitory substances, but also due to co-elution of extracellular DNA, which potentially leads to an overestimate of the actual diversity. Previous studies suggested that eDNA might play an important role in biogeochemical element cycling, horizontal gene transfer and stabilization of biofilm structures. Several protocols based on the precipitation of eDNA e.g. with CTAB and ethanol have already been published. However, using these methods we did not succeed in quantifying very low amounts of eDNA (e.g. <1?g eDNA/g dry wt) in marine sediment even when using DNA carriers like glycogen. Since the recovery of eDNA by precipitation strongly depends on its concentration, these previously published procedures are not adequate for deep biosphere sediment due to the low eDNA content. We have focused on the question whether eDNA could be a source of nitrogen and phosphorus for microbes in the subseafloor biosphere. Therefore we developed a new method for the (semi)-quantitative extraction of eDNA from sediment. The new extraction procedure is based on sequential washing of the sediment to remove simultaneously eDNA and microbial cells without lysing them. After separation of the cells by centrifugation, the eDNA was extracted from the supernatant and purified by adsorption onto a solid phase, followed by removal of the solids and subsequent elution of the pure eDNA. Intracellular DNA (iDNA) was extracted and purified from the cell pellet using a commercial DNA extraction kit. Additional to a very low detection limit and reproducible quantification, this new method allows separation and purification of both extracellular and intracellular DNA to an extent that inhibitors are removed and downstream applications like PCR can be performed. To evaluate the new extraction method two sediments with rather opposing composition were analyzed. Sediment from the South Pacific Gyre, the most oligotrophic oceanic region on earth and organic-rich Baltic Sea sediment (Northern Germany) were processed. Using this new procedure high purity genomic iDNA and eDNA with a molecular size range between 20 bp and 50k bp can be simultaneously recovered even from very oligotrophic sediment with very low cell abundances. The main fraction of recovered eDNA was suitable for downstream applications like PCR and had a molecular size that indicates minimal shearing. Despite about two decades of research many questions about deep subsurface life remain unanswered. The fact that microbes can be found even in deep oligotrophic marine sediment raises the fundamental questions of the types and availability of substrates and their biogeochemical cycling. This is the first study that provides evidence that eDNA is an important potential substrate for microorganisms in the deep biosphere. Also, our results show a link between cell counts and eDNA content, indicating that the eDNA pool in the investigated sediment consist mainly of microbial DNA. Comparative sequence analysis of extracted iDNA and eDNA will provide deeper insights into the origin and turnover of eDNA and the apparent microbial community composition in the deep biosphere.

Alawi, M.; Kallmeyer, J.

2012-12-01

353

Evaluation of DNA Extraction Techniques for Detecting Mycobacterium tuberculosis Complex Organisms in Asian Elephant Trunk Wash Samples?  

PubMed Central

Rapid and sensitive diagnostic assays for the detection of tuberculous mycobacteria in elephants are lacking. DNA extraction with PCR analysis is useful for tuberculosis screening in many species but has not been validated on elephant trunk wash samples. We estimated the analytical sensitivity and specificity of three DNA extraction methods to detect Mycobacterium tuberculosis complex organisms in trunk wash specimens. A ZR soil microbe DNA kit (ZR) and a traditional salt and ethanol precipitation (TSEP) approach were evaluated under three different treatment conditions: heat treatment, phenol treatment, and contamination with Mycobacterium avium. A third approach, using a column filtration method, was evaluated for samples contaminated with soil. Trunk wash samples from uninfected elephants were spiked with various concentrations of M. bovis cells and subjected to the described treatment conditions prior to DNA extraction. Extracted DNA was amplified using IS6110-targeted PCR analysis. The ZR and TSEP methods detected as low as 1 to 5 M. bovis cells and 10 M. bovis cells, respectively, per 1.5 ml of trunk wash under all three conditions. Depending on the amount of soil present, the column filtration method detected as low as 5 to 50 M. bovis cells per 1.5 ml of trunk wash. Analytical specificity was assessed by DNA extraction from species of nontuberculous mycobacteria and amplification using the same PCR technique. Only M. bovis DNA was amplified, indicating 100% analytical specificity of this PCR technique. Our results indicate that these DNA extraction techniques offer promise as useful tests for detection of M. tuberculosis complex organisms in elephant trunk wash specimens.

Kay, Meagan K.; Linke, Lyndsey; Triantis, Joni; Salman, M. D.; Larsen, R. Scott

2011-01-01

354

Smoking Related Carcinogen-DNA Adducts in Biopsy Samples of Human Urinary Bladder: Identification of N-(Deoxyguanosin-8-yl)-4Aminobiphenyl as a Major Adduct  

Microsoft Academic Search

The prevalence of covalent modifications to DNA (carcinogen-DNA adducts) in 42 human urinary bladder biopsy samples was investigated by 32P-postlabeling methods, with enhancement by both nuclease P1 treatment and 1-butanol extraction. Total mean carcinogen-DNA adduct levels and the mean levels of several specific adducts were significantly elevated in DNA samples of 13 current smokers, as opposed to 9 never smokers

Glenn Talaska; Amer Z. S. S. Al-Juburi; Fred F. Kadlubar

1991-01-01

355

Effects of ascorbic acid on sperm motility, viability, acrosome reaction and DNA integrity in teratozoospermic samples.  

PubMed

Background: Oxidative stress in teratozoospermic semen samples caused poor assisted reproductive techniques (ART) outcomes. Among antioxidants, ascorbic acid is a naturally occurring free radical scavenger and as such its presence assists various other mechanisms in decreasing numerous disruptive free radical processes. Objective: The main goal of this study was to evaluate potential protective effects of ascorbic acid supplementation during in vitro culture of teratozoospermic specimens. Materials and Methods: Teratozoospermic semen samples that collected from 15 volunteers were processed, centrifuged and incubated at 37(o)C until sperm swimmed-up. Supernatant was divided into four groups and incubated at 37(o)C for one hour under different experimental conditions: Control, 10 µm A23187, 600µm ascorbic acid and 10 µm A23187+600 µm ascorbic acid. After incubation sperm motility, viability, acrosome reaction, DNA damage and malondialdehyde levels were evaluated. Results: Our results indicated that after one hour incubation, ascorbic acid significantly reduced malondialdehyde level in ascorbic acid group (1.4±0.11 nmol/ml) compared to control group (1.58±0.13 nmol/ml) (p<0.001). At the end of incubation, progressive motility and viability in ascorbic acid group (64.5±8.8% and 80.3±6.4%, respectively) were significantly (p<0.05 and p<0.001, respectively) higher than the control group (54.5±6.8% and 70.9±7.3%, respectively). A23187 significantly (p<0.0001) increased acrosome reaction in A23187 group (37.3±5.6%) compared to control group (8.5±3.2%) and this effect of A23187 attenuated by ascorbic acid in ascorbic acid+A23187 group (17.2±4.4%). DNA fragmentation in ascorbic acid group (20±4.1%) was significantly (p<0.001) lower than controls (28.9±4.6%). Conclusion: In vitro ascorbic acid supplementation during teratozoospermic semen processing for ART could protect teratozoospermic specimens against oxidative stress, and it could improve ART outcome. PMID:24799867

Fanaei, Hamed; Khayat, Samira; Halvaei, Iman; Ramezani, Vahid; Azizi, Yaser; Kasaeian, Amir; Mardaneh, Jalal; Parvizi, Mohammad Reza; Akrami, Maryam

2014-02-01

356

Effects of ascorbic acid on sperm motility, viability, acrosome reaction and DNA integrity in teratozoospermic samples  

PubMed Central

Background: Oxidative stress in teratozoospermic semen samples caused poor assisted reproductive techniques (ART) outcomes. Among antioxidants, ascorbic acid is a naturally occurring free radical scavenger and as such its presence assists various other mechanisms in decreasing numerous disruptive free radical processes. Objective: The main goal of this study was to evaluate potential protective effects of ascorbic acid supplementation during in vitro culture of teratozoospermic specimens. Materials and Methods: Teratozoospermic semen samples that collected from 15 volunteers were processed, centrifuged and incubated at 37oC until sperm swimmed-up. Supernatant was divided into four groups and incubated at 37oC for one hour under different experimental conditions: Control, 10 µm A23187, 600µm ascorbic acid and 10 µm A23187+600 µm ascorbic acid. After incubation sperm motility, viability, acrosome reaction, DNA damage and malondialdehyde levels were evaluated. Results: Our results indicated that after one hour incubation, ascorbic acid significantly reduced malondialdehyde level in ascorbic acid group (1.4±0.11 nmol/ml) compared to control group (1.58±0.13 nmol/ml) (p<0.001). At the end of incubation, progressive motility and viability in ascorbic acid group (64.5±8.8% and 80.3±6.4%, respectively) were significantly (p<0.05 and p<0.001, respectively) higher than the control group (54.5±6.8% and 70.9±7.3%, respectively). A23187 significantly (p<0.0001) increased acrosome reaction in A23187 group (37.3±5.6%) compared to control group (8.5±3.2%) and this effect of A23187 attenuated by ascorbic acid in ascorbic acid+A23187 group (17.2±4.4%). DNA fragmentation in ascorbic acid group (20±4.1%) was significantly (p<0.001) lower than controls (28.9±4.6%). Conclusion: In vitro ascorbic acid supplementation during teratozoospermic semen processing for ART could protect teratozoospermic specimens against oxidative stress, and it could improve ART outcome.

Fanaei, Hamed; Khayat, Samira; Halvaei, Iman; Ramezani, Vahid; Azizi, Yaser; Kasaeian, Amir; Mardaneh, Jalal; Parvizi, Mohammad Reza; Akrami, Maryam

2014-01-01

357

Anaerobic degradation of halogenated benzoic acids coupled to denitrification observed in a variety of sediment and soil samples  

Microsoft Academic Search

Denitrifying enrichment cultures utilizing monochlorinated benzoic acids as a carbon source were established using sediments and soils from a variety of sources as inocula. Enrichment cultures from most of the sites readily degraded 3- and 4chlorobenzoate within 2–4 weeks. Upon refeeding, 3- and 4-chlorobenzoate were rapidly depleted, and stable denitrifying cultures were obtained by repeated dilution and refeeding of the

Max M. Häggblom; Maria D. Rivera; Lily Y. Young

1996-01-01

358

Development of a rapid and efficient method for non-lethal DNA sampling and genotyping in scallops.  

PubMed

Non-lethal DNA sampling has long appealed to researchers studying population and conservation genetics, as it does not necessitate removing individuals permanently from their natural environment or destroying valuable samples. However, such an approach has not yet been well established in bivalves. In this study, we demonstrate that the gill represents a good source of tissue for non-lethal sampling in scallops. Removal of a few gill filaments caused no noticeable behavioral abnormalities or increased mortality rates in Zhikong scallop (Chlamys farreri) during a three-month period of observation. To facilitate rapid gill-based DNA extraction, six methods (MA-MF) were designed and evaluated, each requiring less than one hour of processing time. The optimal method was identified as MF, in terms of maintaining DNA integrity and genotyping accuracy. Further optimization of MF method by orthogonal experimental design suggested that the utilization of gills could be limited to 2 mg of sample, which is sufficient for performing up to 20,000 PCR reactions. We also demonstrate the excellent cross-species utility of MF in two additional scallop species, Yesso scallop (Patinopecten yessoensis) and bay scallop (Argopecten irradians). Taken together, our study provides a rapid and efficient approach for applying non-lethal DNA sampling in bivalve species, which would serve as a valuable tool for maintaining bivalve populations and conservation genetics, as well as in breeding studies. PMID:23874509

Mao, Junxia; Lv, Jia; Miao, Yan; Sun, Changsen; Hu, Liping; Zhang, Ru; Fu, Xiaoteng; Zhang, Lingling; Hu, Xiaoli; Wang, Shi; Bao, Zhenmin

2013-01-01

359

Oral administration of copper to rats leads to increased lymphocyte cellular DNA degradation by dietary polyphenols: implications for a cancer preventive mechanism.  

PubMed

To account for the observed anticancer properties of plant polyphenols, we have earlier proposed a mechanism which involves the mobilization of endogenous copper ions by polyphenols leading to the generation of reactive oxygen species (ROS) that serve as proximal DNA cleaving agents and lead to cell death. Over the last decade we have proceeded to validate our hypothesis with considerable success. As a further confirmation of our hypothesis, in this paper we first show that oral administration of copper to rats leads to elevated copper levels in lymphocytes. When such lymphocytes with a copper overload were isolated and treated with polyphenols EGCG, genistein and resveratrol, an increased level of DNA breakage was observed. Further, preincubation of lymphocytes having elevated copper levels with the membrane permeable copper chelator neocuproine, resulted in inhibition of polyphenol induced DNA degradation. However, membrane impermeable chelator of copper bathocuproine, as well as iron and zinc chelators were ineffective in causing such inhibition in DNA breakage, confirming the involvement of endogenous copper in polyphenol induced cellular DNA degradation. It is well established that serum and tissue concentrations of copper are greatly increased in various malignancies. In view of this fact, the present results further confirm our earlier findings and strengthen our hypothesis that an important anticancer mechanism of plant polyphenols could be the mobilization of intracellular copper leading to ROS-mediated cellular DNA breakage. In this context, it may be noted that cancer cells are under considerable oxidative stress and increasing such stress to cytotoxic levels could be a successful anticancer approach. PMID:21717118

Khan, Husain Y; Zubair, Haseeb; Ullah, Mohd F; Ahmad, Aamir; Hadi, Sheikh M

2011-12-01

360

Blood puncture as a nondestructive sampling tool to obtain DNA in frogs: comparison of protocols and survival analysis.  

PubMed

In molecular biology studies of Anura, nondestructive methods to obtain genetic material are needed as alternatives to toe clipping. This work evaluates a nondestructive method for sampling DNA from blood puncture, comparing the performance of three different extraction protocols (Qiagen Kit, Salting-out and Chelex). We collected 134 individuals of Eleutherodactylus johnstonei, extracting blood via puncture of the medial vein using commercial-grade glucometer lancets. We extracted 100-1880 ng DNA, finding no differences between the extraction protocols. We compared the quality of the resulting DNA through amplification and sequencing of the 16S mitochondrial gene. Amplification was successful for the three extraction protocols, although Chelex showed better performance, making it the most recommendable protocol for extraction of DNA from blood. The resulting sequences corresponded to those registered in the GenBank for this species. Additionally, we found no significant differences in survival or weight change between the individuals that were manipulated and a control group (mean survival 66.7% treated, 62.9% untreated). Data reveal that blood samples obtained by puncture are a convenient alternative to other tissues (phalange, buccal swab, liver) that have traditionally been used as DNA sources for anurans. The technique is applicable to small and large species, covering most anuran diversity, provides enough DNA for many genetic applications and produces no noticeable effect on the survival or performance, given that it does not affect the motor parts or the dexterity of the animals. PMID:22240248

Mendoza, A M; García-Ramírez, J C; Cárdenas-Henao, H

2012-05-01

361

Detection of herpesvirus DNA by the polymerase chain reaction (PCR) in vitreous samples from patients with necrotising retinitis  

PubMed Central

Aims—Viral uveitis and retinitis, usually caused by herpesviruses, are common in immunosuppressed patients. The diagnosis of viral anterior uveitis and retinitis is usually clinical. The polymerase chain reaction (PCR) has been used for the diagnosis of some viral infections, especially those caused by herpesviruses. This paper reports the use of PCR in the diagnosis of viral retinitis in vitreous samples from Brazilian patients. Methods—PCR was used for the diagnosis of necrotising retinitis in vitreous samples from patients from the Hospital São Geraldo, Universidade Federal de Minas Gerais, Brazil. The vitreous samples were collected by paracentesis and stored until analysis. Samples were analysed by PCR using specific primers designed to amplify herpes simplex virus 1 (HSV-1), varicella zoster virus (VZV), or human cytomegalovirus (HCMV). In a case of anterior uveitis, PCR was performed with a sample from the anterior chamber. Results—Herpesvirus DNA was amplified in 11 of 17 samples. HCVM DNA was detected in nine samples but DNA from HSV-1 and VZV were detected only once each. Conclusion—These results strongly suggest that PCR could be used for a rapid complementary diagnosis of viral uveitis and retinitis. A prospective study to evaluate the PCR results, clinical evolution, and treatment is imperative to corroborate the real value of PCR in diagnosis and how it could help the clinicians' approach. Key Words: polymerase chain reaction • uveitis • retinitis

Nogueira, M; Siqueira, R; Freitas, N; Amorim, J; Bonjardim, C; Ferreira, P; Orefice, F; Kroon, E

2001-01-01

362

Comparison of five commercial DNA extraction kits for the recovery of Francisella tularensis DNA from spiked soil samples  

Microsoft Academic Search

Francisella tularensis is the etiologic agent of the zoonotic disease tularemia and is thought to be maintained in the environment principally by various terrestrial and aquatic vertebrate animals. The organism is known to persist in water or mud for long periods of time and Francisella-specific DNA has been identified from water and soil. To gain a better understanding of the

Chris A. Whitehouse; Hannah E. Hottel

2007-01-01

363

A Column Experiment To Determine Black Shale Degradation And Colonization By Means of ?13C and 14C Analysis Of Phospholipid Fatty Acids And DNA Extraction  

NASA Astrophysics Data System (ADS)

We investigated the degradation of black shale organic matter by microbial communities. We inoculated two columns respectively, with the fungi Schizophyllum commune, the gram-positive bacterium Pseudomonas putida and the gram-negative bacteria Streptomyces griseus and Streptomyces chartreusis. These microorganisms are known to degrade a wide variety of organic macromolecules. Additionally, we had two sets of control columns. To one set the same nutrient solution was added as to the inoculated columns and to the other set only sterile deionised water was supplied. All columns contained 1.5 kg of freshly crushed not autoclaved black shale material with a particle size of 0.63-2 mm. The columns were incubated at 28° C and 60% humidity in the dark. The aim was to investigate, which microorganisms live on black shales and if these microorganisms are able to degrade ancient organic matter. We used compound specific stable isotope measurement techniques and compound specific 14C-dating methods. After 183 days PLFAs were extracted from the columns to investigate the microbial community, furthermore we extracted on one hand total-DNA of column material and on the other hand DNA from pure cultures isolates which grew on Kinks-agar B, Starch-casein-nitrate-agar (SCN) and on complete-yeast-medium-agar (CYM). According to the PLFA analysis bacteria dominated in the columns, whereas in pure cultures more fungi were isolated. A principal component analysis revealed differences between the columns in accordance with the inoculation, but it seems that the inoculated microorganisms were replaced by the natural population. For AMS measurements palmitic acid (C 16:0) was re-isolated from total-PLFA-extract with a preparative fraction collector (PFC). Preliminary results of the study revealed that microorganisms are able to degrade black shale material and that PLFA analysis are useful methods to be combined with analysis of stable isotope and 14C measurements to study microbial degradation processes.

Seifert, A.; Gleixner, G.

2008-12-01

364

Simultaneous assessment of the macrobiome and microbiome in a bulk sample of tropical arthropods through DNA metasystematics.  

PubMed

Conventional assessments of ecosystem sample composition are based on morphology-based or DNA barcode identification of individuals. Both approaches are costly and time-consuming, especially when applied to the large number of specimens and taxa commonly included in ecological investigations. Next-generation sequencing approaches can overcome the bottleneck of individual specimen isolation and identification by simultaneously sequencing specimens of all taxa in a bulk mixture. Here we apply multiple parallel amplification primers, multiple DNA barcode markers, 454-pyrosequencing, and Illumina MiSeq sequencing to the same sample to maximize recovery of the arthropod macrobiome and the bacterial and other microbial microbiome of a bulk arthropod sample. We validate this method with a complex sample containing 1,066 morphologically distinguishable arthropods from a tropical terrestrial ecosystem with high taxonomic diversity. Multiamplicon next-generation DNA barcoding was able to recover sequences corresponding to 91% of the distinguishable individuals in a bulk environmental sample, as well as many species present as undistinguishable tissue. 454-pyrosequencing was able to recover 10 more families of arthropods and 30 more species than did conventional Sanger sequencing of each individual specimen. The use of other loci (16S and 18S ribosomal DNA gene regions) also added the detection of species of microbes associated with these terrestrial arthropods. This method greatly decreases the time and money necessary to perform DNA-based comparisons of biodiversity among ecosystem samples. This methodology opens the door to much cheaper and increased capacity for ecological and evolutionary studies applicable to a wide range of socio-economic issues, as well as a basic understanding of how the world works. PMID:24808136

Gibson, Joel; Shokralla, Shadi; Porter, Teresita M; King, Ian; van Konynenburg, Steven; Janzen, Daniel H; Hallwachs, Winnie; Hajibabaei, Mehrdad

2014-06-01

365

Simultaneous assessment of the macrobiome and microbiome in a bulk sample of tropical arthropods through DNA metasystematics  

PubMed Central

Conventional assessments of ecosystem sample composition are based on morphology-based or DNA barcode identification of individuals. Both approaches are costly and time-consuming, especially when applied to the large number of specimens and taxa commonly included in ecological investigations. Next-generation sequencing approaches can overcome the bottleneck of individual specimen isolation and identification by simultaneously sequencing specimens of all taxa in a bulk mixture. Here we apply multiple parallel amplification primers, multiple DNA barcode markers, 454-pyrosequencing, and Illumina MiSeq sequencing to the same sample to maximize recovery of the arthropod macrobiome and the bacterial and other microbial microbiome of a bulk arthropod sample. We validate this method with a complex sample containing 1,066 morphologically distinguishable arthropods from a tropical terrestrial ecosystem with high taxonomic diversity. Multiamplicon next-generation DNA barcoding was able to recover sequences corresponding to 91% of the distinguishable individuals in a bulk environmental sample, as well as many species present as undistinguishable tissue. 454-pyrosequencing was able to recover 10 more families of arthropods and 30 more species than did conventional Sanger sequencing of each individual specimen. The use of other loci (16S and 18S ribosomal DNA gene regions) also added the detection of species of microbes associated with these terrestrial arthropods. This method greatly decreases the time and money necessary to perform DNA-based comparisons of biodiversity among ecosystem samples. This methodology opens the door to much cheaper and increased capacity for ecological and evolutionary studies applicable to a wide range of socio-economic issues, as well as a basic understanding of how the world works.

Gibson, Joel; Shokralla, Shadi; Porter, Teresita M.; King, Ian; van Konynenburg, Steven; Janzen, Daniel H.; Hallwachs, Winnie; Hajibabaei, Mehrdad

2014-01-01

366

Photocatalytic degradation of 4-nitrophenol in aqueous suspension by using polycrystalline TiO 2 samples impregnated with Cu(II)-phthalocyanine  

Microsoft Academic Search

In this paper, the preparation of polycrystalline TiO2 samples impregnated with a modified Cu(II)-phthalocyanine (TiO2–CuPc) is reported along with an investigation on the photocatalytic behavior of this system compared with bare TiO2 (both in the anatase and rutile form) and with TiO2 samples impregnated with not functionalized commercial phthalocyanine (TiO2–CuPc) or with metal free phthalocyanine (TiO2–Pc). The photocatalytic degradation of

Giuseppe Mele; Giuseppe Ciccarella; Giuseppe Vasapollo; Elisa Garc??a-López; Leonardo Palmisano; Mario Schiavello

2002-01-01

367

Bacterial Populations in Samples of Bioleached Copper Ore as Revealed by Analysis of DNA Obtained before and after Cultivation  

Microsoft Academic Search

ThecompositionofbacterialpopulationsincopperbioleachingsystemswasinvestigatedbyanalysisofDNA obtained either directly from ores or leaching solutions or after laboratory cultures. This analysis consisted of thecharacterizationofthespacerregionsbetweenthe16and23SgenesinthebacterialrRNAgeneticlociafter PCR amplification. The sizes of the spacer regions, amplified from DNAs obtained from samples, were comparedwiththesizesofthoseobtainedfromculturesofthemainbacterialspeciesisolatedfrombioleaching systems. This allowed a preliminary assessment of the bacterial species present in the samples. Identification of the bacteria was achieved by partial sequencing of the 16S

EUGENIA JEDLICKI; OMAR ORELLANA; JAIME ROMERO; ANDROMILIO T. ESPEJO

1996-01-01

368

Mitochondrial DNA in a population of individuals from the City of São Paulo. DNA extraction from head and pubic hair and blood  

Microsoft Academic Search

In biological samples with low copy DNA or DNA degraded samples, such as hair and pubic hair, there is a greater probability of obtaining a mitochondrial DNA profile. Hair and pubic hair are often found at crime scenes; in cases of sexual violence is common to find the suspect's pubic hair in the victim's body. This study analyzed, blood, head

C. D. Godoy; I. S. Kunii; K. S. Funabashi; J. M. Cerutti; M. L. A. P. O. Sousa; D. M. Sinagawa; J. A. Soares-Vieira; E. S. M. Iwamura

369

[Comparison of quantitative hepatitis B virus DNA levels in serum and liver biopsy samples of chronic hepatitis B patients].  

PubMed

Chronic hepatitis B (CHB) is an important health problem worldwide. Relapses in CHB patients, even treated, create a major problem in patient management. The aims of this study were the detection of quantitative levels of hepatitis B virus (HBV) DNA in the sera and liver biopsy specimens of CHB patients and the comparison of the results. A total of 69 patients (49 male, 20 female; age range: 19-68 years, mean age: 36.91+11.03 years) who were prediagnosed as CHB in the Infectious Diseases and Clinical Microbiology Department of Kocaeli University (northwestern region of Turkey) were included to this prospective study. HBV-DNA levels of all of the patients were initially measured in the routine laboratory of our hospital by using a commercial real-time polymerase chain reaction (RQ-PCR; iCycler IQ System, BioRad Laboratories) and the mean HBV-DNA level (Serum DNA #2) was detected as 5.6E+6 +/- 8.5E+6 copies/mL. The mean level of ALT in CHB patients was 79.59 +/- 69.7. In our study the HBV-DNA levels of the serum and liver biopsy specimens were also measured by using an "in-house" real-time PCR (RT-PCR) (Techne Quantica, London, UK). Nucleic acid extractions were performed with the use of QIAamp DNA Mini Kit (Qiagen Inc, Hilden, Germany) according to the manufacturer's recommendations. The primers were specific for the X gene region of HBV (forward primer: 5'-TTCGCTTCACCTCTGCACG-3', reverse primer: 5'-CCCAACTCCCAGTCTTTAA-3'; probe: 5'-AATGTCAACGACCGACCTT GAGGCA-pBHQ-3'). Statistical analysis was carried out with Spearman rank analysis. With the use of "in-house" real-time PCR, mean levels of HBV-DNA were found to be 8.99E+6 +/- 3.1E+7 copies/mL in the liver biopsy samples, and 4E+6 +/- 4.4E+6 copies/ mL in serum samples (Serum DNA 1). There were significant positive correlations between the results obtained from in-house (Serum DNA 1) and commercial RT-PCR (Serum DNA 2) (r = 0.300; p = 0.024) methods. Although HBV-DNA levels in the liver tissues were found higher than those in serum samples, no correlation between the HBV-DNA levels of liver biopsy and serum samples (both serum DNA 1 and 2) was detected. This result was attributed to the standardization problems of in-house RT-PCR. In conclusion, liver biopsies performed at the beginning of the therapy to detect the grade of chronic liver disease, would also be searched by means of quantitative HBV-DNA levels, however, since the determination of HBV-DNA load in liver tissues may be difficult, standardized sensitive methods should be used for this purpose. PMID:18697426

Rüzgar, Mehtap; Cetin Akhan, Sila; Vahabo?lu, Haluk

2008-04-01

370

Fungal DNA detected in blood samples of patients who received contaminated methylprednisolone injections reveals increased complexity of causative agents.  

PubMed

Using Exserohilum rostratum-specific and panfungal real-time PCR, we studied 24 blood samples and 2 synovial fluid specimens from 20 patients with persistent or worsening pain following injections of contaminated methylprednisolone. Seven blood specimens from 6 patients were significantly positive for fungal DNA by panfungal PCR, with multiple fungal species identified. PMID:24719442

Zhao, Yanan; Armeanu, Emilian; DiVerniero, Richard; Lewis, Terri A; Dobson, Richard C; Kontoyiannis, Dimitrios P; Roilides, Emmanuel; Walsh, Thomas J; Perlin, David S

2014-06-01

371

Development of DNA Extraction and PCR Amplification Protocols for Detection of Mycoplasma bovis Directly from Milk Samples  

Microsoft Academic Search

Cremonesi, P., Vimercati, C., Pisoni, G., Perez, G., Miranda Ribera, A., Castiglioni, B., Luzzana, M., Ruffo, G. and Moroni,\\u000a P., 2007. Development of DNA extraction and PCR amplification protocols for detection of Mycoplasma bovis directly from milk samples. Veterinary Research Communications, 31(Suppl. 1), 225–227

P. Cremonesi; C. Vimercati; G. Pisoni; G. Perez; A. Miranda Ribera; B. Castiglioni; M. Luzzana; G. Ruffo; P. Moroni

2007-01-01

372

Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic Device  

Microsoft Academic Search

Copy Number Variations (CNVs) of regions of the human genome have been associated with multiple diseases. We present an algorithm which is mathematically sound and computationally efficient to accurately analyze CNV in a DNA sample utilizing a nanofluidic device, known as the digital array. This numerical algorithm is utilized to compute copy number variation and the associated statistical confidence interval

Simant Dube; Jian Qin; Ramesh Ramakrishnan

2008-01-01

373

Mitochondrial DNA Marker EST00083 Is Not Associated with High vs. Average IQ in a German Sample.  

ERIC Educational Resources Information Center

Tested the association of a mitochondrial DNA marker (EST00083) with high IQ in a sample of 47 German adults with high IQ scores and 77 adults with IQs estimated at lower than 110. Results do not support the hypothesis that high IQ is associated with this marker. (SLD)

Moises, Hans W.; Yang, Liu; Kohnke, Michael; Vetter, Peter; Neppert, Jurgen; Petrill, Stephen A.; Plomin, Robert

1998-01-01

374

Rapid Method for Screening Dried Blood Samples on Filter Paper for Human Immunodeficiency Virus Type 1 DNA  

PubMed Central

PCR is a highly sensitive method for the detection of human immunodeficiency virus type 1 (HIV-1) nucleic acids in blood mononuclear cells and plasma. However, blood separation techniques require extensive laboratory support systems and are difficult when a limited volume of blood is available, which is often the case for infants. The use of blood samples stored on filter paper has many advantages for the detection of perinatal HIV-1 infection, but current methods require extraction and purification of target DNA prior to PCR amplification. We report a highly sensitive and rapid method for the extraction and detection of HIV-1 DNA in infant blood samples stored on filter papers. Because this rapid protocol does not involve steps for the removal of potential inhibitors of the PCR, the highest sensitivity is achieved by testing the filter paper lysate in quadruplicate. Assays for HIV-1 DNA were done by using nested PCR techniques that amplify HIV-1 gag DNA from blood spot samples on filter paper and from corresponding viably frozen mononuclear cells separated from venous blood samples obtained from 111 infants born to HIV-1-seropositive mothers. PCR results with blood from filter papers showed 100% specificity (95% confidence internal [CI] 93.1 to 100%) and 96% (95% CI, 88.65 to 98.9%) and 88% (95% CI, 79.2 to 94.5%) sensitivity (for quadruplicate and duplicate tests, respectively) compared to PCR results with blood mononuclear cells. Moreover, this method could detect HIV-1 sequences of multiple subtypes.

Panteleeff, Dana DeVange; John, Grace; Nduati, Ruth; Mbori-Ngacha, Dorothy; Richardson, Barbra; Kreiss, Joan; Overbaugh, Julie

1999-01-01

375

Comparison of DNA Extraction Methods for Microbial Community Profiling with an Application to Pediatric Bronchoalveolar Lavage Samples  

Microsoft Academic Search

Barcoded amplicon sequencing is rapidly becoming a standard method for profiling microbial communities, including the human respiratory microbiome. While this approach has less bias than standard cultivation, several steps can introduce variation including the type of DNA extraction method used. Here we assessed five different extraction methods on pediatric bronchoalveolar lavage (BAL) samples and a mock community comprised of nine

Dana Willner; Joshua Daly; David Whiley; Keith Grimwood; Claire E. Wainwright; Philip Hugenholtz

2012-01-01

376

Effects of the most common methods for the enhancement of latent fingerprints on DNA extraction from forensic samples  

Microsoft Academic Search

The aim of the research was to understand if the use of chemicals compounds used to enhance latent fingerprints, might interfere with the extraction and amplification of DNA from biological samples on crime scenes. Only three methods were used: powders (black and white ones, used on non porous surfaces, and here applied on glass), cyanoacrylate (used on non porous surfaces,

S. Gino; M. Omedei

377

Sera from mice immunized with DNA encoding Porphyromonas gingivalis catalytic or adhesin part of HRgpA inhibit degradation of human fibronectin by HRgpA.  

PubMed

Gingipains are potent virulence factors of Porphyromonas gingivalis and are likely to be associated with the development of periodontitis. It is, therefore, suggested that gingipain inhibition by vaccination could be a useful therapy for adult periodontitis. This study investigated the ability of antibodies raised against the catalytic part and the adhesin/haemagglutinin part of HRgpA to prevent haemagglutination and fibronectin degradation caused by P. gingivalis. We constructed two DNA vaccines, one containing the adhesin part of HRgpA and one with the catalytic part of HRgpA. BALB/c mice were immunized intramuscularly with either catalytic-part-encoding plasmids, adhesin-part-encoding plasmids or empty control plasmids. Sera from mice immunized with the catalytic vaccine or the adhesin vaccine each showed inhibition of human fibronectin degradation. A DNA vaccine encoding the adhesin or catalytic part of HRgpA induces responses that inhibit the degradation of molecules important for the structure and function of gingival and bone tissues. PMID:17241170

Vågnes, K S; Vågnes, O; Bakken, V

2007-02-01

378

Upscaled CTAB-Based DNA Extraction and Real-Time PCR Assays for Fusarium culmorum and F. graminearum DNA in Plant Material with Reduced Sampling Error  

PubMed Central

Fusarium graminearum Schwabe (Gibberella zeae Schwein. Petch.) and F. culmorum W.G. Smith are major mycotoxin producers in small-grain cereals afflicted with Fusarium head blight (FHB). Real-time PCR (qPCR) is the method of choice for species-specific, quantitative estimation of fungal biomass in plant tissue. We demonstrated that increasing the amount of plant material used for DNA extraction to 0.5–1.0 g considerably reduced sampling error and improved the reproducibility of DNA yield. The costs of DNA extraction at different scales and with different methods (commercial kits versus cetyltrimethylammonium bromide-based protocol) and qPCR systems (doubly labeled hybridization probes versus SYBR Green) were compared. A cost-effective protocol for the quantification of F. graminearum and F. culmorum DNA in wheat grain and maize stalk debris based on DNA extraction from 0.5–1.0 g material and real-time PCR with SYBR Green fluorescence detection was developed.

Brandfass, Christoph; Karlovsky, Petr

2008-01-01

379

Hematopoietic gene promoters subjected to a group-combinatorial study of DNA samples: identification of a megakaryocytic selective DNA signature  

PubMed Central

Identification of common sub-sequences for a group of functionally related DNA sequences can shed light on the role of such elements in cell-specific gene expression. In the megakaryocytic lineage, no one single unique transcription factor was described as linage specific, raising the possibility that a cluster of gene promoter sequences presents a unique signature. Here, the megakaryocytic gene promoter group, which consists of both human and mouse 5? non-coding regions, served as a case study. A methodology for group-combinatorial search has been implemented as a customized software platform. It extracts the longest common sequences for a group of related DNA sequences and allows for single gaps of varying length, as well as double- and multiple-gap sequences. The results point to common DNA sequences in a group of genes that is selectively expressed in megakaryocytes, and which does not appear in a large group of control, random and specific sequences. This suggests a role for a combination of these sequences in cell-specific gene expression in the megakaryocytic lineage. The data also point to an intrinsic cross-species difference in the organization of 5? non-coding sequences within the mammalian genomes. This methodology may be used for the identification of regulatory sequences in other lineages.

Hazony, Yehonathan; Lu, Jun; St. Hilaire, Cynthia; Ravid, Katya

2006-01-01