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Sample records for degraded dna samples

  1. Balancing sample accumulation and DNA degradation rates to optimize noninvasive genetic sampling of sympatric carnivores.

    PubMed

    Lonsinger, Robert C; Gese, Eric M; Dempsey, Steven J; Kluever, Bryan M; Johnson, Timothy R; Waits, Lisette P

    2015-07-01

    Noninvasive genetic sampling, or noninvasive DNA sampling (NDS), can be an effective monitoring approach for elusive, wide-ranging species at low densities. However, few studies have attempted to maximize sampling efficiency. We present a model for combining sample accumulation and DNA degradation to identify the most efficient (i.e. minimal cost per successful sample) NDS temporal design for capture-recapture analyses. We use scat accumulation and faecal DNA degradation rates for two sympatric carnivores, kit fox (Vulpes macrotis) and coyote (Canis latrans) across two seasons (summer and winter) in Utah, USA, to demonstrate implementation of this approach. We estimated scat accumulation rates by clearing and surveying transects for scats. We evaluated mitochondrial (mtDNA) and nuclear (nDNA) DNA amplification success for faecal DNA samples under natural field conditions for 20 fresh scats/species/season from <1-112 days. Mean accumulation rates were nearly three times greater for coyotes (0.076 scats/km/day) than foxes (0.029 scats/km/day) across seasons. Across species and seasons, mtDNA amplification success was ≥95% through day 21. Fox nDNA amplification success was ≥70% through day 21 across seasons. Coyote nDNA success was ≥70% through day 21 in winter, but declined to <50% by day 7 in summer. We identified a common temporal sampling frame of approximately 14 days that allowed species to be monitored simultaneously, further reducing time, survey effort and costs. Our results suggest that when conducting repeated surveys for capture-recapture analyses, overall cost-efficiency for NDS may be improved with a temporal design that balances field and laboratory costs along with deposition and degradation rates.

  2. An enzyme-based DNA preparation method for application to forensic biological samples and degraded stains.

    PubMed

    Lounsbury, Jenny A; Coult, Natalie; Miranian, Daniel C; Cronk, Stephen M; Haverstick, Doris M; Kinnon, Paul; Saul, David J; Landers, James P

    2012-09-01

    Extraction of DNA from forensic samples typically uses either an organic extraction protocol or solid phase extraction (SPE) and these methods generally involve numerous sample transfer, wash and centrifugation steps. Although SPE has been successfully adapted to the microdevice, it can be problematic because of lengthy load times and uneven packing of the solid phase. A closed-tube enzyme-based DNA preparation method has recently been developed which uses a neutral proteinase to lyse cells and degrade proteins and nucleases [14]. Following a 20 min incubation of the buccal or whole blood sample with this proteinase, DNA is polymerase chain reaction (PCR)-ready. This paper describes the optimization and quantitation of DNA yield using this method, and application to forensic biological samples, including UV- and heat-degraded whole blood samples on cotton or blue denim substrates. Results demonstrate that DNA yield can be increased from 1.42 (±0.21)ng/μL to 7.78 (±1.40)ng/μL by increasing the quantity of enzyme per reaction by 3-fold. Additionally, there is a linear relationship between the amount of starting cellular material added and the concentration of DNA in the solution, thereby allowing DNA yield estimations to be made. In addition, short tandem repeat (STR) profile results obtained using DNA prepared with the enzyme method were comparable to those obtained with a conventional SPE method, resulting in full STR profiles (16 of 16 loci) from liquid samples (buccal swab eluate and whole blood), dried buccal swabs and bloodstains and partial profiles from UV or heat-degraded bloodstains on cotton or blue denim substrates. Finally, the DNA preparation method is shown to be adaptable to glass or poly(methyl methacrylate) (PMMA) microdevices with little impact on STR peak height but providing a 20-fold reduction in incubation time (as little as 60 s), leading to a ≥1 h reduction in DNA preparation time.

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

    PubMed

    Coble, Michael D

    2012-01-01

    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.

  4. Degradation in forensic trace DNA samples explored by massively parallel sequencing.

    PubMed

    Hanssen, Eirik Nataas; Lyle, Robert; Egeland, Thore; Gill, Peter

    2017-03-01

    Routine forensic analysis using STRs will fail if the DNA is too degraded. The DNA degradation process in biological stain material is not well understood. In this study we sequenced old semen and blood stains by massively parallel sequencing. The sequence data coverage was used to measure degradation across the genome. The results supported the contention that degradation is uniform across the genome, showing no evidence of regions with increased or decreased resistance towards degradation. Thus the lack of genetic regions robust to degradation removes the possibility of using such regions to further optimize analysis performance for degraded DNA.

  5. Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples.

    PubMed

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

    2008-01-01

    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 repeat region. These candidate loci were initially screened based on their small amplicon sizes and locations on chromosomes currently unoccupied by the 13 CODIS STR loci or at least 50 Mb away from them on the same chromosome. They were sequenced and evaluated across more than 600 samples, and their population statistics were determined. The heterozygosities of the new loci were compared with those of the 13 CODIS loci and all were found to be comparable. Only five of the new loci had lower values than the CODIS loci; however, all of these were much smaller in size. This data suggests that these 26 miniSTR loci will serve as useful complements to the CODIS loci to aid in the forensic analysis of degraded DNA, as well as missing persons work and parentage testing with limited next-of-kin reference samples.

  6. Time-dependent RNA degradation affecting cDNA array quality in spontaneous canine tumours sampled using standard surgical procedures.

    PubMed

    Von Euler, Henrik; Khoshnoud, Reza; He, Qimin; Khoshnoud, Aida; Fornander, Tommy; Rutqvist, Lars-Erik; Skog, Sven

    2005-12-01

    Heterogeneous gene expression in tumours and the degradation of RNA when sampling under non-RNAse-free conditions may limit the potential benefit of cDNA array studies. This study examines changes in the integrity of RNA by means of RNA gel electrophoresis at various post-operative intervals on canine mammary tumours (n=10) and malignant lymphoma (n=1). The tumours were cut into pieces (3-5 mm diameter, approximately 50 mg) and kept in tubes without RNAse-free buffer at room temperature. No special precautions were taken to avoid the influences of Rnase; rather, normal surgical procedures were used. We found that total RNA of the mammary tumours started to degrade within 30 min of the operation, and the rate of degradation increased up to 4 h, which was the last time point included in this study. RNA in the lymphoma tumours degraded more rapidly, and was completely degraded at 30 min post-operation. The degradation of mRNA in the mammary tumours, as studied by human cDNA arrays, was heterogeneous, i.e. some mRNA degraded completely, some only partially. This indicates that the mRNA degradation rate varied depending on the type of mRNA. However, since we found that gene expression differs depending on the part of the mammary tumour examined, one cannot exclude that the variation in the mRNA degradation rate may simply reflect heterogeneous gene expression within the tumour. We conclude that RNA integrity is unaffected immediately after sampling under non-RNAse-free conditions; however, the tumour sample should be preserved under RNAse-free conditions within 15 min to avoid RNA degradation. This is a much shorter time interval than previously reported in other similar studies; however, these studies generally treated normal tissue, under which 3-5 h non-RNAse-free conditions have been found not to affect RNA quality.

  7. DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification

    PubMed Central

    2013-01-01

    Background Mitochondrial DNA (mtDNA) typing can be a useful aid for identifying people from compromised samples when nuclear DNA is too damaged, degraded or below detection thresholds for routine short tandem repeat (STR)-based analysis. Standard mtDNA typing, focused on PCR amplicon sequencing of the control region (HVS I and HVS II), is limited by the resolving power of this short sequence, which misses up to 70% of the variation present in the mtDNA genome. Methods We used in-solution hybridisation-based DNA capture (using DNA capture probes prepared from modern human mtDNA) to recover mtDNA from post-mortem human remains in which the majority of DNA is both highly fragmented (<100 base pairs in length) and chemically damaged. The method ‘immortalises’ the finite quantities of DNA in valuable extracts as DNA libraries, which is followed by the targeted enrichment of endogenous mtDNA sequences and characterisation by next-generation sequencing (NGS). Results We sequenced whole mitochondrial genomes for human identification from samples where standard nuclear STR typing produced only partial profiles or demonstrably failed and/or where standard mtDNA hypervariable region sequences lacked resolving power. Multiple rounds of enrichment can substantially improve coverage and sequencing depth of mtDNA genomes from highly degraded samples. The application of this method has led to the reliable mitochondrial sequencing of human skeletal remains from unidentified World War Two (WWII) casualties approximately 70 years old and from archaeological remains (up to 2,500 years old). Conclusions This approach has potential applications in forensic science, historical human identification cases, archived medical samples, kinship analysis and population studies. In particular the methodology can be applied to any case, involving human or non-human species, where whole mitochondrial genome sequences are required to provide the highest level of maternal lineage discrimination

  8. MiniSTR multiplex systems based on non-CODIS loci for analysis of degraded DNA samples.

    PubMed

    Asamura, H; Fujimori, S; Ota, M; Fukushima, H

    2007-11-15

    We describe two short amplicon autosomal short tandem repeat (miniSTR) quadruplex systems for eight loci D1S1171, D2S1242, D3S1545, D4S2366, D12S391, D16S3253, D20S161, and D21S1437, unlinked from the combined DNA index system (non-CODIS) loci, using newly designed primer sets. The results of an assay of 411 Japanese individuals showed that polymerase chain reaction (PCR) products within the eight loci were less than 150bp in size, without the seven additional bases for adenylation. The frequency distributions in the loci showed no deviations from Hardy-Weinberg equilibrium expectations. The accumulated power of discrimination and power of exclusion for the eight loci were 0.9999999991 and 0.998, respectively. For assay of highly degraded DNA, including artificially degraded samples and the degraded forensic casework samples assessed with the present miniSTR quadruplex systems, the systems proved quite effective in analyzing degraded DNA.

  9. Evaluating the interaction of faecal pellet deposition rates and DNA degradation rates to optimize sampling design for DNA-based mark-recapture analysis of Sonoran pronghorn.

    PubMed

    Woodruff, S P; Johnson, T R; Waits, L P

    2015-07-01

    Knowledge of population demographics is important for species management but can be challenging in low-density, wide-ranging species. Population monitoring of the endangered Sonoran pronghorn (Antilocapra americana sonoriensis) is critical for assessing the success of recovery efforts, and noninvasive DNA sampling (NDS) could be more cost-effective and less intrusive than traditional methods. We evaluated faecal pellet deposition rates and faecal DNA degradation rates to maximize sampling efficiency for DNA-based mark-recapture analyses. Deposition data were collected at five watering holes using sampling intervals of 1-7 days and averaged one pellet pile per pronghorn per day. To evaluate nuclear DNA (nDNA) degradation, 20 faecal samples were exposed to local environmental conditions and sampled at eight time points from one to 124 days. Average amplification success rates for six nDNA microsatellite loci were 81% for samples on day one, 63% by day seven, 2% by day 14 and 0% by day 60. We evaluated the efficiency of different sampling intervals (1-10 days) by estimating the number of successful samples, success rate of individual identification and laboratory costs per successful sample. Cost per successful sample increased and success and efficiency declined as the sampling interval increased. Results indicate NDS of faecal pellets is a feasible method for individual identification, population estimation and demographic monitoring of Sonoran pronghorn. We recommend collecting samples <7 days old and estimate that a sampling interval of four to seven days in summer conditions (i.e., extreme heat and exposure to UV light) will achieve desired sample sizes for mark-recapture analysis while also maximizing efficiency [Corrected].

  10. Forensic genetic SNP typing of low-template DNA and highly degraded DNA from crime case samples.

    PubMed

    Børsting, Claus; Mogensen, Helle Smidt; Morling, Niels

    2013-05-01

    Heterozygote imbalances leading to allele drop-outs and disproportionally large stutters leading to allele drop-ins are known stochastic phenomena related to STR typing of low-template DNA (LtDNA). The large stutters and the many drop-ins in typical STR stutter positions are artifacts from the PCR amplification of tandem repeats. These artifacts may be avoided by typing bi-allelic markers instead of STRs. In this work, the SNPforID multiplex assay was used to type LtDNA. A sensitized SNP typing protocol was introduced, that increased signal strengths without increasing noise and without affecting the heterozygote balance. Allele drop-ins were only observed in experiments with 25 pg of DNA and not in experiments with 50 and 100 pg of DNA. The allele drop-in rate in the 25 pg experiments was 0.06% or 100 times lower than what was previously reported for STR typing of LtDNA. A composite model and two different consensus models were used to interpret the SNP data. Correct profiles with 42-49 SNPs were generated from the 50 and 100 pg experiments, whereas a few incorrect genotypes were included in the generated profiles from the 25 pg experiments. With the strict consensus model, between 35 and 48 SNPs were correctly typed in the 25 pg experiments and only one allele drop-out (error rate: 0.07%) was observed in the consensus profiles. A total of 28 crime case samples were selected for typing with the sensitized SNPforID protocol. The samples were previously typed with old STR kits during the crime case investigation and only partial profiles (0-6 STRs) were obtained. Eleven of the samples could not be quantified with the Quantifiler™ Human DNA Quantification kit because of partial or complete inhibition of the PCR. For eight of these samples, SNP typing was only possible when the buffer and DNA polymerase used in the original protocol was replaced with the AmpFℓSTR(®) SEfiler Plus™ Master Mix, which was developed specifically for challenging forensic samples. All

  11. A quadruplex real-time qPCR assay for the simultaneous assessment of total human DNA, human male DNA, DNA degradation and the presence of PCR inhibitors in forensic samples: a diagnostic tool for STR typing.

    PubMed

    Hudlow, William R; Chong, Mavis Date; Swango, Katie L; Timken, Mark D; Buoncristiani, Martin R

    2008-03-01

    A quadruplex real-time qPCR assay was developed to simultaneously assess total human DNA, human male DNA, DNA degradation and PCR inhibitors in forensic samples. Specifically, the assay utilizes a approximately 170-190bp target sequence that spans the TH01 STR locus to quantify total human DNA (nuTH01), a 137 bp target sequence directly adjacent to the SRY gene to quantify human male DNA (nuSRY), a 67 bp target sequence flanking the CSF1PO STR locus (nuCSF) to assess degradation (nuCSF:nuTH01 ratio) and a 77 bp synthetic DNA template used as an internal PCR control target sequence (IPC) for the assessment of PCR inhibition. Validation studies, performed on an ABI 7500 SDS instrument using TaqMan and TaqManMGB detection, indicate each of the targets in the quadruplex assay performs effectively and is informative even when challenged with DNase-degraded and hematin-inhibited samples. The nuTH01-nuSRY-nuCSF-IPC quadruplex qPCR assay is envisioned to assist in the choice of the most informative DNA typing system available, which may include standard autosomal STR typing when the results indicate the presence of non-degraded, single gender DNA or non-degraded, male:female mixtures at ratios expected to yield probative alleles; Y STR typing in samples containing a male component that is overwhelmed by the presence of an excess of female DNA; reduced amplicon size STR typing ("MiniSTRs") where the nuCSF:nuTH01 ratio indicates the sample is highly degraded; enhanced STR amplification with additional AmpliTaq Gold/BSA and/or sample clean-up when the presence of PCR inhibitors is suggested by a delayed IPC C(T) value or mitochondrial DNA typing in samples where little to no nuclear DNA is detected. The present study includes evaluations of species specificity, sensitivity, precision, reproducibility, male-female mixtures, population samples and applications to various casework-type samples as indicated by the Scientific Working Group on DNA Analysis Methods (SWGDAM

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

    PubMed

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

    2014-11-01

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

  13. Mini-midi-mito: adapting the amplification and sequencing strategy of mtDNA to the degradation state of crime scene samples.

    PubMed

    Berger, Cordula; Parson, Walther

    2009-06-01

    The degradation state of some biological traces recovered from the crime scene requires the amplification of very short fragments to attain a useful mitochondrial (mt)DNA sequence. We have previously introduced two mini-multiplex assays that amplify 10 overlapping control region (CR) fragments in two separate multiplex PCRs, which brought successful CR consensus sequences from even highly degraded DNA extracts. This procedure requires a total of 20 sequencing reactions per sample, which is laborious and cost intensive. For only moderately degraded samples that we encounter more frequently with typical mtDNA casework material, we developed two new multiplex assays that use a subset of the mini-amplicon primers but embrace larger fragments (midis) and require only 10 sequencing reactions to build a double-stranded CR consensus sequence. We used a preceding mtDNA quantitation step by real-time PCR with two different target fragments (143 and 283 bp) that roughly correspond to the average fragment sizes of the different multiplex approaches to estimate size-dependent mtDNA quantities and to aid the choice of the appropriate PCR multiplexes with respect to quality of the results and required costs.

  14. 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

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

    2014-07-01

    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.

  15. Authentication of forensic DNA samples.

    PubMed

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

    2010-02-01

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

  16. DNA Profiling Success Rates from Degraded Skeletal Remains in Guatemala.

    PubMed

    Johnston, Emma; Stephenson, Mishel

    2016-07-01

    No data are available regarding the success of DNA Short Tandem Repeat (STR) profiling from degraded skeletal remains in Guatemala. Therefore, DNA profiling success rates relating to 2595 skeletons from eleven cases at the Forensic Anthropology Foundation of Guatemala (FAFG) are presented. The typical postmortem interval was 30 years. DNA was extracted from bone powder and amplified using Identifiler and Minifler. DNA profiling success rates differed between cases, ranging from 50.8% to 7.0%, the overall success rate for samples was 36.3%. The best DNA profiling success rates were obtained from femur (36.2%) and tooth (33.7%) samples. DNA profiles were significantly better from lower body bones than upper body bones (p = <0.0001). Bone samples from males gave significantly better profiles than samples from females (p = <0.0001). These results are believed to be related to bone density. The findings are important for designing forensic DNA sampling strategies in future victim recovery investigations.

  17. Quantifiler® Trio Kit and forensic samples management: a matter of degradation.

    PubMed

    Vernarecci, Stefano; Ottaviani, Enrica; Agostino, Alessandro; Mei, Elisabetta; Calandro, Lisa; Montagna, Paola

    2015-05-01

    DNA collected from crime scenes may have experienced different levels of degradation. This is mainly due to sample exposure to different environmental factors. The impact of DNA degradation on short tandem repeat (STR) profiling can lead to partial or null information and in some cases, the identification of the trace may fail. The availability of a system enabling the assessment not only of the quantity of the DNA but also of its quality in terms of degradation would result in shorter time for sample processing, more reliable identifications and cost reduction by predicting the quality of the DNA profiles prior to STR analysis. We report here a study on 181 selected degraded DNA samples extracted from real crime scene evidence. The selected samples were processed by combining the use of a new commercial quantification kit (Quantifiler® Trio) with a new 24 marker multiplex PCR amplification kit (Globalfiler® Kit). Applying different statistical analyses we investigated the reliability of the Degradation Index provided by the Quantifiler® Trio in determining the level of DNA degradation in a forensic sample. This useful information can be used to predict the quality of the profile obtained after STR amplification. The combination of such a quantification kit with different PCR protocols allowed us to define practical guidelines for processing degraded forensic DNA samples with a simplified and comprehensive approach.

  18. Radiation-induced degradation of DNA bases

    NASA Astrophysics Data System (ADS)

    Douki, T.; Delatour, T.; Martini, R.; Cadet, J.

    1999-01-01

    Radio-induced degradation of DNA involves radical processes. A series of lesions among the major bases degradation products has been measured in isolated DNA exposed to gamma radiation in aerated aqueous solution. Degradation can be accounted for by the formation of hydroxyl radicals upon radiolysis of water (indirect effect). The four bases are degraded in high yield. Direct effect has been mimicked by photo-induced electron abstraction from the bases producing their radical cation. Quantification of the modified bases showed that guanine is the preferential target. This can be explained by its lower oxidation potential and charge transfer phenomena. La décomposition radio-induite de l'ADN fait intervenir des processus radicalaires. Une série de lésions choisies parmi les produits majeurs de dégradation des bases a été mesurée dans de l'ADN isolé exposé au rayonnement en solution aqueuse aérée. Les modifications sont alors dues aux radicaux hydroxyles produits par la radiolyse de l'eau (effet indirect) et les quatre bases sont efficacement dégradées. L'arrachement d'électrons aux bases par photosensibilisation pour produire leur radical cation, a été utilisé comme modèle de l'effet direct. La quantification des bases modifiées montre que la guanine est préférentiellement dégradée. Cette observation peut s'expliquer par le plus faible potentiel d'oxydation de cette base ainsi que par les phénomènes de transfert de charge vers les guanines.

  19. Survival and DNA degradation in anhydrobiotic tardigrades.

    PubMed

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

    2009-12-01

    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.

  20. Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase.

    PubMed

    Gansauge, Marie-Theres; Gerber, Tobias; Glocke, Isabelle; Korlević, Petra; Lippik, Laurin; Nagel, Sarah; Riehl, Lara Maria; Schmidt, Anna; Meyer, Matthias

    2017-01-23

    DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3' biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude.

  1. New insights from old bones: DNA preservation and degradation in permafrost preserved mammoth remains.

    PubMed

    Schwarz, Carsten; Debruyne, Regis; Kuch, Melanie; McNally, Elizabeth; Schwarcz, Henry; Aubrey, Andrew D; Bada, Jeffrey; Poinar, Hendrik

    2009-06-01

    Despite being plagued by heavily degraded DNA in palaeontological remains, most studies addressing the state of DNA degradation have been limited to types of damage which do not pose a hindrance to Taq polymerase during PCR. Application of serial qPCR to the two fractions obtained during extraction (demineralization and protein digest) from six permafrost mammoth bones and one partially degraded modern elephant bone has enabled further insight into the changes which endogenous DNA is subjected to during diagenesis. We show here that both fractions exhibit individual qualities in terms of the prevailing type of DNA (i.e. mitochondrial versus nuclear DNA) as well as the extent of damage, and in addition observed a highly variable ratio of mitochondrial to nuclear DNA among the six mammoth samples. While there is evidence suggesting that mitochondrial DNA is better preserved than nuclear DNA in ancient permafrost samples, we find the initial DNA concentration in the bone tissue to be as relevant for the total accessible mitochondrial DNA as the extent of DNA degradation post-mortem. We also evaluate the general applicability of indirect measures of preservation such as amino-acid racemization, bone crystallinity index and thermal age to these exceptionally well-preserved samples.

  2. New insights from old bones: DNA preservation and degradation in permafrost preserved mammoth remains

    PubMed Central

    Schwarz, Carsten; Debruyne, Regis; Kuch, Melanie; McNally, Elizabeth; Schwarcz, Henry; Aubrey, Andrew D.; Bada, Jeffrey; Poinar, Hendrik

    2009-01-01

    Despite being plagued by heavily degraded DNA in palaeontological remains, most studies addressing the state of DNA degradation have been limited to types of damage which do not pose a hindrance to Taq polymerase during PCR. Application of serial qPCR to the two fractions obtained during extraction (demineralization and protein digest) from six permafrost mammoth bones and one partially degraded modern elephant bone has enabled further insight into the changes which endogenous DNA is subjected to during diagenesis. We show here that both fractions exhibit individual qualities in terms of the prevailing type of DNA (i.e. mitochondrial versus nuclear DNA) as well as the extent of damage, and in addition observed a highly variable ratio of mitochondrial to nuclear DNA among the six mammoth samples. While there is evidence suggesting that mitochondrial DNA is better preserved than nuclear DNA in ancient permafrost samples, we find the initial DNA concentration in the bone tissue to be as relevant for the total accessible mitochondrial DNA as the extent of DNA degradation post-mortem. We also evaluate the general applicability of indirect measures of preservation such as amino-acid racemization, bone crystallinity index and thermal age to these exceptionally well-preserved samples. PMID:19321502

  3. 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...

  4. Microfluidic DNA sample preparation method and device

    DOEpatents

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

    2002-01-01

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

  5. Leaf tissue sampling and DNA extraction protocols.

    PubMed

    Semagn, Kassa

    2014-01-01

    Taxonomists must be familiar with a number of issues in collecting and transporting samples using freezing methods (liquid nitrogen and dry ice), desiccants (silica gel and blotter paper), and preservatives (CTAB, ethanol, and isopropanol), with each method having its own merits and limitations. For most molecular studies, a reasonably good quality and quantity of DNA is required, which can only be obtained using standard DNA extraction protocols. There are many DNA extraction protocols that vary from simple and quick ones that yield low-quality DNA but good enough for routine analyses to the laborious and time-consuming standard methods that usually produce high quality and quantities of DNA. The protocol to be chosen will depend on the quality and quantity of DNA needed, the nature of samples, and the presence of natural substances that may interfere with the extraction and subsequent analysis. The protocol described in this chapter has been tested for extracting DNA from eight species and provided very good quality and quantity of DNA for different applications, including those genotyping methods that use restriction enzymes.

  6. [DNA degradation during standard alkaline of thermal denaturation].

    PubMed

    Drozhdeniuk, A P; Sulimova, G E; Vaniushin, B F

    1976-01-01

    Essential degradation 8 DNA (up to 10 per cent) with liberation of acid-soluble fragments takes place on the standard alkaline (0,01 M sodium phosphate, pH 12, 60 degrees, 15 min) or thermal (0.06 M sodium phosphate buffer, pH 6.8, 102 degrees C, 15 min) denaturation. This degradation is more or less selective: fraction of low molecular weight fragments, isolated by hydroxyapatite cromatography and eluted by 0.06 M sodium phosphate buffer, pH 6.8 is rich in adenine and thymine and contains about 2 times less 5-methylcytosine than the total wheat germ DNA. The degree of degradation of DNA on thermal denaturation is higher than on alkaline degradation. Therefore while studying reassociation of various DNA, one and the same standard method of DNA denaturation should be used. Besides, both the level of DNA degradation and the nature of the resulting products (fragments) should be taken into account.

  7. Flow cytometric detection method for DNA samples

    DOEpatents

    Nasarabadi,Shanavaz; Langlois, Richard G.; Venkateswaran, Kodumudi S.

    2011-07-05

    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.

  8. Flow cytometric detection method for DNA samples

    DOEpatents

    Nasarabadi, Shanavaz; Langlois, Richard G.; Venkateswaran, Kodumudi S.

    2006-08-01

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2005-05-01

    In this project, a new set of multiplexed PCR reactions has been developed for the analysis of degraded DNA. These DNA markers, known as Miniplexes, utilize primers that have shorter amplicons for use in short tandem repeat (STR) analysis of degraded DNA. In our work we have defined six of these new STR multiplexes, each of which consists of 3 to 4 reduced size STR loci, and each labeled with a different fluorescent dye. When compared to commercially available STR systems, reductions in size of up to 300 basepairs are possible. In addition, these newly designed amplicons consist of loci that are fully compatible with the the national computer DNA database known as CODIS. To demonstrate compatibility with commercial STR kits, a concordance study of 532 DNA samples of Caucasian, African American, and Hispanic origin was undertaken There was 99.77% concordance between allele calls with the two methods. Of these 532 samples, only 15 samples showed discrepancies at one of 12 loci. These occurred predominantly at 2 loci, vWA and D13S317. DNA sequencing revealed that these locations had deletions between the two primer binding sites. Uncommon deletions like these can be expected in certain samples and will not affect the utility of the Miniplexes as tools for degraded DNA analysis. The Miniplexes were also applied to enzymatically digested DNA to assess their potential in degraded DNA analysis. The results demonstrated a greatly improved efficiency in the analysis of degraded DNA when compared to commercial STR genotyping kits. A series of human skeletal remains that had been exposed to a variety of environmental conditions were also examined. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only sixteen percent of the samples tested generated full profiles with a commercial kit. In addition, complete profiles were obtained for eleven of the twelve Miniplex loci which had amplicon size ranges less than 200 base pairs

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

    PubMed Central

    Sun, Weimin; Sun, Xiaoxu

    2012-01-01

    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. PMID:22327600

  11. Electrostatic sampling of trace DNA from clothing.

    PubMed

    Zieger, Martin; Defaux, Priscille Merciani; Utz, Silvia

    2016-05-01

    During acts of physical aggression, offenders frequently come into contact with clothes of the victim, thereby leaving traces of DNA-bearing biological material on the garments. Since tape-lifting and swabbing, the currently established methods for non-destructive trace DNA sampling from clothing, both have their shortcomings in collection efficiency and handling, we thought about a new collection method for these challenging samples. Testing two readily available electrostatic devices for their potential to sample biological material from garments made of different fabrics, we found one of them, the electrostatic dust print lifter (DPL), to perform comparable to well-established sampling with wet cotton swabs. In simulated aggression scenarios, we had the same success rate for the establishment of single aggressor profiles, suitable for database submission, with both the DPL and wet swabbing. However, we lost a substantial amount of information with electrostatic sampling, since almost no mixed aggressor-victim profiles suitable for database entry could be established, compared to conventional swabbing. This study serves as a proof of principle for electrostatic DNA sampling from items of clothing. The technique still requires optimization before it might be used in real casework. But we are confident that in the future it could be an efficient and convenient contribution to the toolbox of forensic practitioners.

  12. Species identification of protected carpet pythons suitable for degraded forensic samples.

    PubMed

    Ciavaglia, Sherryn; Donnellan, Stephen; Henry, Julianne; Linacre, Adrian

    2014-09-01

    In this paper we report on the identification of a section of mitochondrial DNA that can be used to identify the species of protected and illegally traded pythons of the genus Morelia. Successful enforcement of wildlife laws requires forensic tests that can identify the species nominated in the relevant legislation. The potentially degraded state of evidentiary samples requires that forensic investigation using molecular genetic species identification is optimized to interrogate small fragments of DNA. DNA was isolated from 35 samples of Morelia spilota from which the complete cytochrome b was sequenced. The ND6 gene was also sequenced in 32 of these samples. Additional DNA sequences were generated from 9 additional species of Morelia. The sequences were aligned by Geneious and imported into MEGA to create phylogenetic trees based on the entire complex of approximately 1,706 base pairs (bp). To mimic degraded DNA, which is usually found in forensic cases, short sub-sections of the full alignment were used to generate phylogenetic trees. The sub-sections that had the greatest DNA sequence information were in parts of the cytochrome b gene. Our results highlight that legislation is presently informed by inadequate taxonomy. We demonstrated that a 278 bp region of the cytochrome b gene recovered the topology of the phylogenetic tree found with the entire gene sequence and correctly identified species of Morelia with a high degree of confidence. The locus described in this report will assist in the successful prosecution of alleged illegal trade in python species.

  13. Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples.

    PubMed Central

    Sayler, G S; Shields, M S; Tedford, E T; Breen, A; Hooper, S W; Sirotkin, K M; Davis, J W

    1985-01-01

    The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined. The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained. The colony hybridization technique allowed detection of one colony containing TOL plasmid among 10(6) Escherichia coli colonies of nonhomologous DNA. Comparisons between population estimates derived from growth on selective substrates and from hybridizations were examined. Findings indicated that standard sole carbon source enumeration procedures for degradative populations lead to overestimations due to nonspecific growth of other bacteria on the microcontaminant carbon sources present in the media. Population estimates based on the selective growth of a microcosm population on two aromatic substrates (toluene and naphthalene) and estimates derived from DNA-DNA colony hybridizations, using the TOL or NAH plasmid as a probe, corresponded with estimates of substrate mineralization rates and past exposure to environmental contaminants. The applications of such techniques are hoped to eventually allow enumeration of any specific gene sequences in the environment, including both anabolic and catabolic genes. In addition, this procedure should prove useful in monitoring recombinant DNA clones released into environmental situations. Images PMID:4004244

  14. Improved purification and PCR amplification of DNA from environmental samples.

    PubMed

    Arbeli, Ziv; Fuentes, Cilia L

    2007-07-01

    Purification and PCR amplification procedures for DNA extracted from environmental samples (soil, compost, and river sediment) were improved by introducing three modifications: precipitation of DNA with 5% polyethylene glycol 8000 (PEG) and 0.6 M NaCl; filtration with a Sepharose 4B-polyvinylpolypyrrolidone (PVPP) spin column; and addition of skim milk (0.3% w/v) to the PCR reaction solution. Humic substances' concentration after precipitation with 5% PEG was 2.57-, 5.3-, and 78.9-fold lower than precipitation with 7.5% PEG, 10% PEG, and isopropanol, respectively. After PEG precipitation, Sepharose, PVPP and the combined (Sepharose-PVPP) column removed 92.3%, 89.5%, and 98%, respectively, of the remaining humic materials. Each of the above-mentioned modifications improved PCR amplification of the 16S rRNA gene. DNA extracted by the proposed protocol is cleaner than DNA extracted by a commercial kit. Nevertheless, the improvement of DNA purification did not improve the detection limit of atrazine degradation gene atzA.

  15. 28 CFR 28.12 - Collection of DNA samples.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 1 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 § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a...

  16. 28 CFR 28.12 - Collection of DNA samples.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 1 2011-07-01 2011-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a...

  17. 28 CFR 28.12 - Collection of DNA samples.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 28 Judicial Administration 1 2014-07-01 2014-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a...

  18. 28 CFR 28.12 - Collection of DNA samples.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 28 Judicial Administration 1 2012-07-01 2012-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a...

  19. 28 CFR 28.12 - Collection of DNA samples.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 28 Judicial Administration 1 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 § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a...

  20. Comparison of preprocessing methods and storage times for touch DNA samples

    PubMed Central

    Dong, Hui; Wang, Jing; Zhang, Tao; Ge, Jian-ye; Dong, Ying-qiang; Sun, Qi-fan; Liu, Chao; Li, Cai-xia

    2017-01-01

    Aim To select appropriate preprocessing methods for different substrates by comparing the effects of four different preprocessing methods on touch DNA samples and to determine the effect of various storage times on the results of touch DNA sample analysis. Method Hand touch DNA samples were used to investigate the detection and inspection results of DNA on different substrates. Four preprocessing methods, including the direct cutting method, stubbing procedure, double swab technique, and vacuum cleaner method, were used in this study. DNA was extracted from mock samples with four different preprocessing methods. The best preprocess protocol determined from the study was further used to compare performance after various storage times. DNA extracted from all samples was quantified and amplified using standard procedures. Results The amounts of DNA and the number of alleles detected on the porous substrates were greater than those on the non-porous substrates. The performances of the four preprocessing methods varied with different substrates. The direct cutting method displayed advantages for porous substrates, and the vacuum cleaner method was advantageous for non-porous substrates. No significant degradation trend was observed as the storage times increased. Conclusion Different substrates require the use of different preprocessing method in order to obtain the highest DNA amount and allele number from touch DNA samples. This study provides a theoretical basis for explorations of touch DNA samples and may be used as a reference when dealing with touch DNA samples in case work. PMID:28252870

  1. Degradation of poly(glycoamidoamine) DNA delivery vehicles: polyamide hydrolysis at physiological conditions promotes DNA release.

    PubMed

    Liu, Yemin; Reineke, Theresa M

    2010-02-08

    Poly(glycoamidoamine)s (PGAAs) are a group of efficient and degradable gene delivery vehicles that consist of three main functionalities: carbohydrate groups, secondary amines, and amide bonds. Herein, we have created nonhydroxylated models to these structures by polymerizing oxylate, succinate, or adipate groups with pentaethylenehexamine. The resulting polymers (named O4, S4, and A4, respectively) were created to understand how the absence of hydroxyl groups and changes in the amide bond spacing affect polymer degradation, plasmid DNA (pDNA) complexation, toxicity, and transfection efficiency in vitro. An additional model was also created that retains a galactaramide unit, but we have replaced the secondary amines with ethyleneoxide units (GO2) to understand the effects of the amine groups on polymer degradation. We have found that the secondary amines and hydroxyls are necessary to facilitate rapid degradation of these polymers, and analogues lacking hydroxyls or amines did not degrade over the time course of the study. Through electron-withdrawing and hydrogen bonding, the hydroxyls appear to activate the carbonyls of the amide bond to hydrolysis via an inductive electron withdrawing effect. Through titration experiments, PGAA degradation appears not to affect the polymer buffering capacity. Furthermore, we have found that PGAA degradation may enhance gene expression by releasing pDNA from polyplexes (polymer-pDNA complexes) and, thus, exposing it to undergo transcription and translation. The difference in the optimal pH that promotes degradation of the PGAAs and the hydroxyl-free analogues may prove to be a useful means to achieve pH-regulated DNA release from polyplexes by specifically modulating the chemical structures.

  2. Novel Phenanthrene-Degrading Bacteria Identified by DNA-Stable Isotope Probing.

    PubMed

    Jiang, Longfei; Song, Mengke; Luo, Chunling; Zhang, Dayi; Zhang, Gan

    2015-01-01

    Microorganisms responsible for the degradation of phenanthrene in a clean forest soil sample were identified by DNA-based stable isotope probing (SIP). The soil was artificially amended with either 12C- or 13C-labeled phenanthrene, and soil DNA was extracted on days 3, 6 and 9. Terminal restriction fragment length polymorphism (TRFLP) results revealed that the fragments of 219- and 241-bp in HaeIII digests were distributed throughout the gradient profile at three different sampling time points, and both fragments were more dominant in the heavy fractions of the samples exposed to the 13C-labeled contaminant. 16S rRNA sequencing of the 13C-enriched fraction suggested that Acidobacterium spp. within the class Acidobacteria, and Collimonas spp. within the class Betaproteobacteria, were directly involved in the uptake and degradation of phenanthrene at different times. To our knowledge, this is the first report that the genus Collimonas has the ability to degrade PAHs. Two PAH-RHDα genes were identified in 13C-labeled DNA. However, isolation of pure cultures indicated that strains of Staphylococcus sp. PHE-3, Pseudomonas sp. PHE-1, and Pseudomonas sp. PHE-2 in the soil had high phenanthrene-degrading ability. This emphasizes the role of a culture-independent method in the functional understanding of microbial communities in situ.

  3. Novel Phenanthrene-Degrading Bacteria Identified by DNA-Stable Isotope Probing

    PubMed Central

    Luo, Chunling; Zhang, Dayi; Zhang, Gan

    2015-01-01

    Microorganisms responsible for the degradation of phenanthrene in a clean forest soil sample were identified by DNA-based stable isotope probing (SIP). The soil was artificially amended with either 12C- or 13C-labeled phenanthrene, and soil DNA was extracted on days 3, 6 and 9. Terminal restriction fragment length polymorphism (TRFLP) results revealed that the fragments of 219- and 241-bp in HaeIII digests were distributed throughout the gradient profile at three different sampling time points, and both fragments were more dominant in the heavy fractions of the samples exposed to the 13C-labeled contaminant. 16S rRNA sequencing of the 13C-enriched fraction suggested that Acidobacterium spp. within the class Acidobacteria, and Collimonas spp. within the class Betaproteobacteria, were directly involved in the uptake and degradation of phenanthrene at different times. To our knowledge, this is the first report that the genus Collimonas has the ability to degrade PAHs. Two PAH-RHDα genes were identified in 13C-labeled DNA. However, isolation of pure cultures indicated that strains of Staphylococcus sp. PHE-3, Pseudomonas sp. PHE-1, and Pseudomonas sp. PHE-2 in the soil had high phenanthrene-degrading ability. This emphasizes the role of a culture-independent method in the functional understanding of microbial communities in situ. PMID:26098417

  4. Characterization of degradation and heterozygote balance by simulation of the forensic DNA analysis process.

    PubMed

    Hansson, Oskar; Egeland, Thore; Gill, Peter

    2017-03-01

    Simulation experiments were used to show the impact of varying extraction efficiency, aliquot proportion, and PCR efficiency on the heterozygote balance of a range of diploid and haploid cells. Reducing either parameters introduces variance. It is well-known that the variance in heterozygote balance increases as the amount of DNA is reduced. Surprisingly the distribution is in fact diamond shaped - the variance start to decrease at very low amounts of DNA. Simulations suggest that pristine diluted DNA is an acceptable approximation in validations to infer heterozygote balance. However, the difference in distribution of the variance between diploid and haploid cell types may, under some circumstances, need to be considered in statistical models. Finally, we exemplify how simulations can be used to predict the outcome of PCR for degraded samples. Visualizing the predicted DNA profile as an electropherogram can help to identify the best approach for sample processing.

  5. Degradable starch nanoparticle assisted ethanol precipitation of DNA.

    PubMed

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

    2014-09-22

    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.

  6. DNase-activatable fluorescence probes visualizing the degradation of exogenous DNA in living cells

    NASA Astrophysics Data System (ADS)

    Gong, Ping; Shi, Bihua; Zhang, Pengfei; Hu, Dehong; Zheng, Mingbin; Zheng, Cuifang; Gao, Duyang; Cai, Lintao

    2012-03-01

    This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the occurrence of a red fluorescence signal due to fluorescence resonance energy transfer (FRET). DNase in biological samples was detected using DFProbes and the fluorescence imaging in living cells was performed using DFprobe-modified Au nanoparticles. The results show that DFProbes have good responses to DNase, and can clearly visualize the degradation of exogenous DNA in cells in real time. The well-designed probes might be useful in tracing the dynamic changes of exogenous DNA and nanocarriers in vitro and in vivo.This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the

  7. Kinetics of ethanol degradation in forensic blood samples.

    PubMed

    Ferrari, L A; Triszcz, J M; Giannuzzi, L

    2006-09-12

    To determine ethanol in human post-mortem blood samples is problematic, largely due to the inappropriate and variable methods of preserving and storing, which can cause decomposition and loss of alcohol concentration. In this study, four crucial parameters of sample conservation were studied: temperature (T), percentage of air chamber in container (%CA), ethanol concentration in blood and post-mortem time. Blood samples from post-mortem cases were stored under different conditions (ethanol levels were known in all cases); factorial design variables: (%CA) 0, 5, 20, 35, 65%; storage temperature: 25, 4 and -10 degrees C; in a total of 15 experiments. No preserving agent was used in samples. Quantification of ethanol in blood was carried out by gas chromatography with head-space FID detector. Initial ethanol concentration ranged from 0.50 to 4.30 g/L. The kinetics of degradation observed was pseudo-first-order. The parameter that characterised the kinetics of ethanol degradation (k(0)) ranged from (4 x 10(-4) and 5.0 x 10(-1) day(-1)), depending on storage conditions. A strong dependence between ethanol degradation and the content of the air chamber was observed and this dependence was found to be stronger than that between degradation and temperature; there was an experimental relation between (k(0)) and (%CA). Activation energy for different conditions, i.e. 0, 5, 20, 35 and 65 (%CA), were calculated and contour plots were made. A mathematical equation relating air chamber, temperature and ethanol concentration at a certain time was determined. This equation allowed estimation of initial concentrations of ethanol with minimal error. A good correlation between experimental data and data calculated with the equation was obtained (r(2) = 0.9998). The best storage conditions were: 0% CA and storage at -10 degrees C, obtaining an ethanol degradation of 0.01% after 15 days. However, 33% of ethanol degradation was obtained with 35% CA at 25 degrees C after 15 days. This

  8. The impact of DNA contamination of bone samples in forensic case analysis and anthropological research.

    PubMed

    von Wurmb-Schwark, Nicole; Heinrich, Anke; Freudenberg, Mechthild; Gebühr, Michael; Schwark, Thorsten

    2008-05-01

    Contamination precautions and quality control are great issues when human bones are investigated genetically. This is especially true for historical samples with only minute amounts of usually highly degraded DNA. But also in forensic routine analysis, sometimes DNA has to be isolated from bones in equally bad conditions, e.g. from burned victims. In such cases, there are several eventualities to contaminate the sample with foreign DNA, for example caused by the recovery of the bones, by trace investigation on a crime scene, or - of course - during handling in the lab. We present the investigation of artificially contaminated historical bone samples which contained no original DNA. Three different kind of contamination were studied: (1) touching of the samples, (2) application of saliva, and (3) application of pure DNA. The samples were genetically investigated without and with the employment of a defined cleaning protocol of the bones. The results show that pure DNA can usually not be removed from the bones and that saliva is a similar thread for subsequent DNA analysis. After the cleaning procedure about 70% of saliva contaminated samples still yielded reproducible STR profiles implicating severe problems for the investigation of highly degraded bone fragments. Simple touching of the specimens seems not to be a real problem for genetic investigations since the obtained signals were not reproducible.

  9. Post Mortem DNA Degradation of Human Tissue Experimentally Mummified in Salt

    PubMed Central

    Shved, Natallia; Haas, Cordula; Papageorgopoulou, Christina; Akguel, Guelfirde; Paulsen, Katja; Bouwman, Abigail; Warinner, Christina; Rühli, Frank

    2014-01-01

    Mummified human tissues are of great interest in forensics and biomolecular archaeology. The aim of this study was to analyse post mortem DNA alterations in soft tissues in order to improve our knowledge of the patterns of DNA degradation that occur during salt mummification. In this study, the lower limb of a female human donor was amputated within 24 h post mortem and mummified using a process designed to simulate the salt dehydration phase of natural or artificial mummification. Skin and skeletal muscle were sampled at multiple time points over a period of 322 days and subjected to genetic analysis. Patterns of genomic fragmentation, miscoding lesions, and overall DNA degradation in both nuclear and mitochondrial DNA was assessed by different methods: gel electrophoresis, multiplex comparative autosomal STR length amplification, cloning and sequence analysis, and PCR amplification of different fragment sizes using a damage sensitive recombinant polymerase. The study outcome reveals a very good level of DNA preservation in salt mummified tissues over the course of the experiment, with an overall slower rate of DNA fragmentation in skin compared to muscle. PMID:25337822

  10. Multiple DNA extractions coupled with stable-isotope probing of anthracene-degrading bacteria in contaminated soil.

    PubMed

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

    2011-05-01

    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 (13)C-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-(13)C]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 (13)C-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.

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

    PubMed Central

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

    2011-01-01

    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. PMID:21398486

  12. Difficulties of sex determination from forensic bone degraded DNA: A comparison of three methods.

    PubMed

    Quincey, Danielle; Carle, Georges; Alunni, Véronique; Quatrehomme, Gérald

    2013-09-01

    Sex determination is of paramount importance in forensic anthropology. Numerous anthropological methods have been described, including visual assessments and various measurements of bones. Nevertheless, whatever the method used, the percentage of correct classification of a single bone usually varies between 80% and 95%, due to significant intra- and inter-population variations, and sometimes variations coming from secular trends. DNA is increasingly used in a forensic context. But forensic DNA extraction from bone raises several issues, because the samples are very often badly altered and/or in very small quantity. Nuclear DNA is difficult to get from degraded samples, according to low copy number, at least in comparison with mitochondrial DNA. In a forensic context (as in a paeleoanthropological context) DNA sex determination is usually complicated by the weak amount of DNA, the degraded nature of nucleic acids, the presence of enzymatic inhibitors in DNA extracts, the possible faint amplification of Y band and the risk of contamination during either excavation or manipulation of samples. The aim of this work was to compare three methods of DNA sex determination from bones: procedure #1 using a single PCR amplification, procedure #2 using a double PCR amplification, and procedure #3 adding bleaching for decontamination of the bone, instead of simply rubbing the bone. These processes were applied to samples of bones (49 samples coming from 39 individuals) that were in various states of post mortem alteration. The main results are the following. (i) No DNA could be extracted from three skulls (parietal bones, mastoid process), the compact bone of one rib, and the diaphysis of one femur; (ii) there was a contamination in three skulls; and (iii) the Y band did not appear in two male cases, with one of the three procedures (male tibia, procedure #2) and with procedures #2 and #3 (male femur). This study emphasises the main issue while working with altered bones: the

  13. Assessment of DNA degradation induced by thermal and UV radiation processing: implications for quantification of genetically modified organisms.

    PubMed

    Ballari, Rajashekhar V; Martin, Asha

    2013-12-01

    DNA quality is an important parameter for the detection and quantification of genetically modified organisms (GMO's) using the polymerase chain reaction (PCR). Food processing leads to degradation of DNA, which may impair GMO detection and quantification. This study evaluated the effect of various processing treatments such as heating, baking, microwaving, autoclaving and ultraviolet (UV) irradiation on the relative transgenic content of MON 810 maize using pRSETMON-02, a dual target plasmid as a model system. Amongst all the processing treatments examined, autoclaving and UV irradiation resulted in the least recovery of the transgenic (CaMV 35S promoter) and taxon-specific (zein) target DNA sequences. Although a profound impact on DNA degradation was seen during the processing, DNA could still be reliably quantified by Real-time PCR. The measured mean DNA copy number ratios of the processed samples were in agreement with the expected values. Our study confirms the premise that the final analytical value assigned to a particular sample is independent of the degree of DNA degradation since the transgenic and the taxon-specific target sequences possessing approximately similar lengths degrade in parallel. The results of our study demonstrate that food processing does not alter the relative quantification of the transgenic content provided the quantitative assays target shorter amplicons and the difference in the amplicon size between the transgenic and taxon-specific genes is minimal.

  14. Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine

    PubMed Central

    Chaitanya, Lakshmi; Ralf, Arwin; van Oven, Mannis; Kupiec, Tomasz; Chang, Joseph; Lagacé, Robert

    2015-01-01

    ABSTRACT Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long‐range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications. PMID:26387877

  15. Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine.

    PubMed

    Chaitanya, Lakshmi; Ralf, Arwin; van Oven, Mannis; Kupiec, Tomasz; Chang, Joseph; Lagacé, Robert; Kayser, Manfred

    2015-12-01

    Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylogenetic resolution. Most established protocols on the different massively parallel sequencing (MPS) platforms, however, invariably involve the PCR amplification of large fragments, typically several kilobases in size, which may fail due to mtDNA fragmentation in the available degraded materials. We introduce a MPS tiling approach for simultaneous whole human mt genome sequencing using 161 short overlapping amplicons (average 200 bp) with the Ion Torrent Personal Genome Machine. We illustrate the performance of this new method by sequencing 20 DNA samples belonging to different worldwide mtDNA haplogroups. Additional quality control, particularly regarding the potential detection of nuclear insertions of mtDNA (NUMTs), was performed by comparative MPS analysis using the conventional long-range amplification method. Preliminary sensitivity testing revealed that detailed haplogroup inference was feasible with 100 pg genomic input DNA. Complete mt genome coverage was achieved from DNA samples experimentally degraded down to genomic fragment sizes of about 220 bp, and up to 90% coverage from naturally degraded samples. Overall, we introduce a new approach for whole mt genome MPS analysis from degraded and nondegraded materials relevant to resolve and infer maternal genetic ancestry at complete resolution in anthropological, evolutionary, medical, and forensic applications.

  16. Automated DNA extraction for large numbers of plant samples.

    PubMed

    Mehle, Nataša; Nikolić, Petra; Rupar, Matevž; Boben, Jana; Ravnikar, Maja; Dermastia, Marina

    2013-01-01

    The method described here is a rapid, total DNA extraction procedure applicable to a large number of plant samples requiring pathogen detection. The procedure combines a simple and quick homogenization step of crude extracts with DNA extraction based upon the binding of DNA to magnetic beads. DNA is purified in an automated process in which the magnetic beads are transferred through a series of washing buffers. The eluted DNA is suitable for efficient amplification in PCR reactions.

  17. Identification of Streptococcus parasanguinis DNA contamination in human buccal DNA samples

    PubMed Central

    2013-01-01

    Background The use of buccal swabs in clinical and scientific studies is a very popular method of collecting DNA, due to its non-invasive nature of collection. However, contamination of the DNA sample may interfere with analysis. Findings Here we report the finding of Streptococcus parasanguinis bacterial DNA contamination in human buccal DNA samples, which led to preferential amplification of bacterial sequence with PCR primers designed against human sequence. Conclusion Contamination of buccal-derived DNA with bacterial DNA can be significant, and may influence downstream genetic analysis. One needs to be aware of possible bacterial contamination when interpreting abnormal findings following PCR amplification of buccal swab DNA samples. PMID:24266944

  18. Quantitative analysis of genomic DNA degradation in whole blood under various storage conditions for molecular diagnostic testing.

    PubMed

    Permenter, Jessalyn; Ishwar, Arjun; Rounsavall, Angie; Smith, Maddie; Faske, Jennifer; Sailey, Charles J; Alfaro, Maria P

    2015-12-01

    Proper storage of whole blood is crucial for isolating nucleic acids from leukocytes and to ensure adequate performance of downstream assays in the molecular diagnostic laboratory. Short-term and long-term storage recommendations are lacking for successful isolation of genomic DNA (gDNA). Container type (EDTA or heparin), temperature (4 °C and room temperature) and time (1-130 days) were assessed as criterion for sample acceptance policies. The percentage of integrated area (%Ti) between 150 and 10,000 bp from the 2200 TapeStation electropherogram was calculated to measure gDNA degradation. Refrigerated EDTA samples yielded gDNA with low %Ti (high quality). Heparinized samples stored at room temperature yielded gDNA of worst quality. Downstream analysis demonstrated that the quality of the gDNA correlated with the quality of the data; samples with high %Ti generated significantly lower levels of high molecular weight amplicons. Recommendations from these analyses include storing blood samples intended for nucleic acid isolation in EDTA tubes at 4 °C for long term storage (>10 days). gDNA should be extracted within 3 days when blood is stored at room temperature regardless of the container. Finally, refrigerated heparinized samples should not be stored longer than 9 days if expecting high quality gDNA isolates. Laboratories should consider many factors, in addition to the results obtained herein, to update their policies for sample acceptance for gDNA extraction intended for molecular genetic testing.

  19. Retrospective study of the impact of miniSTRs on forensic DNA profiling of touch DNA samples.

    PubMed

    Van Nieuwerburgh, Filip; Van Hoofstat, David; Van Neste, Christophe; Deforce, Dieter

    2014-09-01

    The theoretical advantages of miniSTRs are undeniable. Several studies show that miniSTRs are more sensitive and robust in the analysis of low template and degraded DNA. In this study we want to show the overall benefit of using miniSTRs in real forensic casework samples and show the percentage of samples that benefit from analysis with additional miniSTR loci in terms of resulting in a useful profile. The considered samples were 3064 touch DNA samples, analyzed in our accredited routine forensic DNA profiling laboratory between mid 2009 and mid 2013. Of these 3064 samples, 618 samples were analyzed using 13 loci, 532 samples using 15 loci and 1914 samples using 20 loci of which 5 were the mini- and midi-STR loci that were added to the extended European Standard Set (ESS). The retrospective results show a small increased success rate after implementation of extra loci and an even smaller increase after the implementation of the mini- and midi-STR analysis. The percentage of touch DNA samples that benefit from the analysis of additional mini- and midi-STR loci is limited.

  20. DNA extraction from rice endosperm (including a protocol for extraction of DNA from ancient seed samples).

    PubMed

    Mutou, Chiaki; Tanaka, Katsunori; Ishikawa, Ryuji

    2014-01-01

    Deoxyribonucleic acid (DNA) extracted from endosperm can be effectively used for rapid genotyping using seed tissue, to evaluate seed quality from packaged grains and to determine the purity of milled grains. Methods outlined here are optimal procedures to isolate DNA from endosperm tissue of modern rice grains and of aged rice remains preserved between 50 and 100 years. The extracted DNA can be used to amplify regions of chloroplast genomic DNA (ctDNA), mitochondrial genomic DNA (mtDNA), and nuclear genomic DNA using standard PCR protocols. In addition, we describe an optimal procedure to process archaeological grain specimens, aged for a couple of thousand years, to isolate DNA from these ancient samples, referred to here as ancient DNA (aDNA). The aDNA can be successfully amplified by PCR using appropriate primer pairs designed specifically for aDNA amplification.

  1. Rapid cleanup of bacterial DNA from samples containing aerosol contaminants

    NASA Astrophysics Data System (ADS)

    Menking, Darrell E.; Kracke, Suzanne K.; Emanuel, Peter A.; Valdes, James J.

    1999-01-01

    Polymerase Chain Reaction (PCR) is an in vitro enzymatic, synthetic method used to amplify specific DNA sequences from organisms. Detection of DNA using gene probes allows for absolute identification not only of specific organisms, but also of genetic material in recombinant organisms. PCR is an exquisite biological method for detecting bacteria in aerosol samples. A major challenge facing detection of DNA from field samples is that they are almost sure to contain impurities, especially impurities that inhibit amplification through PCR. DNA is being extracted from air, sewage/stool samples, food, sputum, a water and sediment; however, multi- step, time consuming methods are required to isolate the DNA from the surrounding contamination. This research focuses on developing a method for rapid cleanup of DNA which combines extraction and purification of DNA while, at the same time, removing inhibitors from 'dirty samples' to produce purified, PCR-ready DNA. GeneReleaser produces PCR-ready DNA in a rapid five-minute protocol. GeneReleaser resin was able to clean up sample contain micrograms of typical aerosol and water contaminants. The advantages of using GR are that it is rapid, inexpensive, requires one-step, uses no hazardous material and produces PCR-ready DNA.

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

    PubMed

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

    2016-11-01

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

  3. DNA identification of Salvia divinorum samples.

    PubMed

    Murphy, Terence M; Bola, Gurpreet

    2013-01-01

    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.

  4. Preservation and rapid purification of DNA from decomposing human tissue samples.

    PubMed

    Sorensen, Amy; Rahman, Elizabeth; Canela, Cassandra; Gangitano, David; Hughes-Stamm, Sheree

    2016-11-01

    One of the key features to be considered in a mass disaster is victim identification. However, the recovery and identification of human remains are sometimes complicated by harsh environmental conditions, limited facilities, loss of electricity and lack of refrigeration. If human remains cannot be collected, stored, or identified immediately, bodies decompose and DNA degrades making genotyping more difficult and ultimately decreasing DNA profiling success. In order to prevent further DNA damage and degradation after collection, tissue preservatives may be used. The goal of this study was to evaluate three customized (modified TENT, DESS, LST) and two commercial DNA preservatives (RNAlater and DNAgard(®)) on fresh and decomposed human skin and muscle samples stored in hot (35°C) and humid (60-70% relative humidity) conditions for up to three months. Skin and muscle samples were harvested from the thigh of three human cadavers placed outdoors for up to two weeks. In addition, the possibility of purifying DNA directly from the preservative solutions ("free DNA") was investigated in order to eliminate lengthy tissue digestion processes and increase throughput. The efficiency of each preservative was evaluated based on the quantity of DNA recovered from both the "free DNA" in solution and the tissue sample itself in conjunction with the quality and completeness of downstream STR profiles. As expected, DNA quantity and STR success decreased with time of decomposition. However, a marked decrease in DNA quantity and STR quality was observed in all samples after the bodies entered the bloat stage (approximately six days of decomposition in this study). Similar amounts of DNA were retrieved from skin and muscle samples over time, but slightly more complete STR profiles were obtained from muscle tissue. Although higher amounts of DNA were recovered from tissue samples than from the surrounding preservative, the average number of reportable alleles from the "free DNA" was

  5. DNA extraction and quantitation of forensic samples using the phenol-chloroform method and real-time PCR.

    PubMed

    Köchl, Silvano; Niederstätter, Harald; Parson, Walther

    2005-01-01

    Forensic laboratories are increasingly confronted with problematic samples from the scene of crime, containing only minute amounts of deoxyribonucleic acid (DNA), which may include polymerase chain reaction (PCR)-inhibiting substances. Efficient DNA extraction procedures, as well as accurate DNA quantification methods, are critical steps involved in the process of successful DNA analysis of such samples. The phenol-chloroform method is a sensitive method for the extraction of DNA from a wide variety of forensic samples, although it is known to be laborious compared with single-tube extraction methods. The relatively high DNA recovery and the quality of the extracted DNA speak for itself. For reliable and sensitive DNA quantitation, the application of real-time PCR is described. We modified a published real-time PCR assay, which allows for the combined analysis of nuclear and mitochondrial DNA, by introducing 1) improved hybridization probes with the use of minor groove binders; 2) an internal positive control (for both nuclear and mitochondrial DNA) for the detection of PCR inhibitors; and 3) different amplicon lengths for the determination of the degradation state of the DNA. The internal positive controls were constructed by site directed mutagenesis by overlap extension of the wild-type mitochondrial and nuclear DNA target with the advantage that no additional probes, which are cost-intensive, are required. The quantitation system is accomplished as a modular concept, which allows for the combined determination of the above-mentioned features (quantity/inhibition or quantity/degradation) depending on the situation.

  6. Co-extraction of DNA and PLFA from soil samples.

    PubMed

    Brewer, Sheridan; Techtmann, Stephen M; Mahmoudi, Nagissa; Niang, Dijibril; Pfiffner, Susan; Hazen, Terry C

    2015-08-01

    Lipid/DNA co-extraction from one sample is attractive in limiting biases associated with microbial community analysis from separate extractions. We sought to enhance established co-extraction methods and use high-throughput 16S rRNA sequencing to identify preferentially extracted taxa from co-extracted DNA. Co-extraction results in low DNA yields and distinct community structure changes.

  7. Getting DNA copy numbers without control samples

    PubMed Central

    2012-01-01

    Background The selection of the reference to scale the data in a copy number analysis has paramount importance to achieve accurate estimates. Usually this reference is generated using control samples included in the study. However, these control samples are not always available and in these cases, an artificial reference must be created. A proper generation of this signal is crucial in terms of both noise and bias. We propose NSA (Normality Search Algorithm), a scaling method that works with and without control samples. It is based on the assumption that genomic regions enriched in SNPs with identical copy numbers in both alleles are likely to be normal. These normal regions are predicted for each sample individually and used to calculate the final reference signal. NSA can be applied to any CN data regardless the microarray technology and preprocessing method. It also finds an optimal weighting of the samples minimizing possible batch effects. Results Five human datasets (a subset of HapMap samples, Glioblastoma Multiforme (GBM), Ovarian, Prostate and Lung Cancer experiments) have been analyzed. It is shown that using only tumoral samples, NSA is able to remove the bias in the copy number estimation, to reduce the noise and therefore, to increase the ability to detect copy number aberrations (CNAs). These improvements allow NSA to also detect recurrent aberrations more accurately than other state of the art methods. Conclusions NSA provides a robust and accurate reference for scaling probe signals data to CN values without the need of control samples. It minimizes the problems of bias, noise and batch effects in the estimation of CNs. Therefore, NSA scaling approach helps to better detect recurrent CNAs than current methods. The automatic selection of references makes it useful to perform bulk analysis of many GEO or ArrayExpress experiments without the need of developing a parser to find the normal samples or possible batches within the data. The method is

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

    PubMed Central

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

    2014-01-01

    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. PMID:24748965

  9. Evidence of degradation of polybrominated biphenyls in soil samples from Michigan

    SciTech Connect

    Hill, R.H. Jr.; Patterson, D.G.; Orti, D.L.; Holler, J.S.; Needham, L.L.; Sirmans, S.L.; Liddle, J.A.

    1982-01-01

    Soil samples obtained from the former polybrominated biphenyls (PBB) manufacturing site in Michigan were analyzed by gas chromatography and gas chromatography with mass spectrometric detection. The results indicate significant degradation of the PBB residue in the soil sample. The soil sample with the highest concentration of PBB had the greatest degree of degradation. Principal degradation products include 2,3', 4,4', 5-pentabromobiphenyl, 2,2', 4,4', 5-pentabromobiphenyl and two unidentified tetrabromobiphenyls. The degradation pattern observed supports a photochemical decomposition mechanism. These degraded residues may be more toxic than the original Firemaster residues. The implications of the results are discussed.

  10. Mode of degradation of plasmid DNA with ozone.

    PubMed

    Sawadaishi, K; Miura, K; Ohthuka, E; Ueda, T; Ishizaki, K; Shinriki, N

    1984-01-01

    The ozonization of pBR322 closed circular DNA showed the conversion to open circular DNA. The damaged site was investigated by restriction mapping. The results showed the damage and subsequent cleavage of the DNA strand of ccDNA by ozonization may occur at the region sensitive to nuclease S1.

  11. Targeted DNA degradation using a CRISPR device stably carried in the host genome

    PubMed Central

    Caliando, Brian J.; Voigt, Christopher A.

    2015-01-01

    Once an engineered organism completes its task, it is useful to degrade the associated DNA to reduce environmental release and protect intellectual property. Here we present a genetically encoded device (DNAi) that responds to a transcriptional input and degrades user-defined DNA. This enables engineered regions to be obscured when the cell enters a new environment. DNAi is based on type-IE CRISPR biochemistry and a synthetic CRISPR array defines the DNA target(s). When the input is on, plasmid DNA is degraded 108-fold. When the genome is targeted, this causes cell death, reducing viable cells by a factor of 108. Further, the CRISPR nuclease can direct degradation to specific genomic regions (for example, engineered or inserted DNA), which could be used to complicate recovery and sequencing efforts. DNAi can be stably carried in an engineered organism, with no impact on cell growth, plasmid stability or DNAi inducibility even after passaging for >2 months. PMID:25988366

  12. Targeted DNA degradation using a CRISPR device stably carried in the host genome.

    PubMed

    Caliando, Brian J; Voigt, Christopher A

    2015-05-19

    Once an engineered organism completes its task, it is useful to degrade the associated DNA to reduce environmental release and protect intellectual property. Here we present a genetically encoded device (DNAi) that responds to a transcriptional input and degrades user-defined DNA. This enables engineered regions to be obscured when the cell enters a new environment. DNAi is based on type-IE CRISPR biochemistry and a synthetic CRISPR array defines the DNA target(s). When the input is on, plasmid DNA is degraded 10(8)-fold. When the genome is targeted, this causes cell death, reducing viable cells by a factor of 10(8). Further, the CRISPR nuclease can direct degradation to specific genomic regions (for example, engineered or inserted DNA), which could be used to complicate recovery and sequencing efforts. DNAi can be stably carried in an engineered organism, with no impact on cell growth, plasmid stability or DNAi inducibility even after passaging for >2 months.

  13. Cytolysis by Ca-permeable transmembrane channels. Pore formation causes extensive DNA degradation and cell lysis

    PubMed Central

    1989-01-01

    This study investigates the effect of the purified membrane pore formers, staphylococcal alpha-toxin and CTL perforin, on target cell lysis as measured by 51Cr release and on nuclear damage as measured by DNA degradation and 125IUdR release. Both pore formers cause dose- dependent cell lysis, which is accompanied by DNA release. The ratio of DNA/Cr release depends on the nature of target cell and shows the same pattern as the ratio of release of the two markers reported for CTL- mediated lysis of the same targets. DNA degradation is dependent on the presence of intracellular Ca in the target cell and is totally blocked if Ca is chelated by Quin 2 intracellularly and EGTA extracellularly. DNA degradation, in addition, is inhibited by the lysosomotropic agents NH4Cl, chloroquine, and monensin. rTNF doubles the degree of DNA degradation mediated by alpha-toxin in 3-h assays. We conclude that pore formers alone can mediate DNA degradation. In addition, they may promote the uptake of other factors and thereby accelerate their time course of action. DNA degradation by pore formers requires active target participation in a pathway that is dependent on intracellular Ca and lysosomes. These aspects of target lysis resemble CTL- and NK cell- mediated cytolysis. PMID:2538546

  14. Conserved primers for DNA barcoding historical and modern samples from New Zealand and Antarctic birds.

    PubMed

    Patel, Selina; Waugh, John; Millar, Craig D; Lambert, David M

    2010-05-01

    Our ability to DNA barcode the birds of the world is based on the effective amplification and sequencing of a 648 base pair (bp) region of the mitochondrial cytochrome c oxidase (COI or cox1) gene. For many geographic regions the large numbers of vouchered specimens necessary for the construction of a DNA barcoding database have already been collected and are available in museums and other institutions. However, many of these specimens are old (>20 years) and are stored as either fixed study skins or dried skeletons. DNA extracted from such historical samples is typically degraded and, generally, only short DNA fragments can be recovered from such specimens making the recovery of the barcoding region as a single fragment difficult. We report two sets of conserved primers that allow the amplification of the entire DNA barcoding region in either three or five overlapping fragments. These primer sets allow the recovery of DNA barcodes from valuable historical specimens that in many cases are unique in that they are unable or unlikely to be collected again. We also report three new primers that in combination allow the effective amplification from modern samples of the entire DNA barcoding region as a single DNA fragment for 17 orders of Southern Hemisphere birds.

  15. Preservation of RNA and DNA from mammal samples under field conditions.

    PubMed

    Camacho-Sanchez, Miguel; Burraco, Pablo; Gomez-Mestre, Ivan; Leonard, Jennifer A

    2013-07-01

    Ecological and conservation genetics require sampling of organisms in the wild. Appropriate preservation of the collected samples, usually by cryostorage, is key to the quality of the genetic data obtained. Nevertheless, cryopreservation in the field to ensure RNA and DNA stability is not always possible. We compared several nucleic acid preservation solutions appropriate for field sampling and tested them on rat (Rattus rattus) blood, ear and tail tip, liver, brain and muscle. We compared the efficacy of a nucleic acid preservation (NAP) buffer for DNA preservation against 95% ethanol and Longmire buffer, and for RNA preservation against RNAlater (Qiagen) and Longmire buffer, under simulated field conditions. For DNA, the NAP buffer was slightly better than cryopreservation or 95% ethanol, but high molecular weight DNA was preserved in all conditions. The NAP buffer preserved RNA as well as RNAlater. Liver yielded the best RNA and DNA quantity and quality; thus, liver should be the tissue preferentially collected from euthanized animals. We also show that DNA persists in nonpreserved muscle tissue for at least 1 week at ambient temperature, although degradation is noticeable in a matter of hours. When cryopreservation is not possible, the NAP buffer is an economical alternative for RNA preservation at ambient temperature for at least 2 months and DNA preservation for at least 10 months.

  16. Developmental validation of the Quantifiler(®) HP and Trio Kits for human DNA quantification in forensic samples.

    PubMed

    Holt, Allison; Wootton, Sharon Chao; Mulero, Julio J; Brzoska, Pius M; Langit, Emanuel; Green, Robert L

    2016-03-01

    The quantification of human genomic DNA is a necessary first step in the DNA casework sample analysis workflow. DNA quantification determines optimal sample input amounts for subsequent STR (short tandem repeat) genotyping procedures, as well as being a useful screening tool to identify samples most likely to provide probative genotypic evidence. To better mesh with the capabilities of newest-generation STR analysis assays, the Quantifiler(®) HP and Quantifiler(®) Trio DNA Quantification Kits were designed for greater detection sensitivity and more robust performance with samples that contain PCR inhibitors or degraded DNA. The new DNA quantification kits use multiplex TaqMan(®) assay-based fluorescent probe technology to simultaneously quantify up to three human genomic targets, allowing samples to be assessed for total human DNA, male contributor (i.e., Y-chromosome) DNA, as well as a determination of DNA degradation state. The Quantifiler HP and Trio Kits use multiple-copy loci to allow for significantly improved sensitivity compared to earlier-generation kits that employ single-copy target loci. The kits' improved performance provides better predictive ability for results with downstream, newest-generation STR assays, and their shortened time-to-result allows more efficient integration into the forensic casework analysis workflow.

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

    PubMed Central

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

    2012-01-01

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

  18. Regulated degradation of chromosome replication proteins DnaA and CtrA in Caulobacter crescentus.

    PubMed

    Gorbatyuk, Boris; Marczynski, Gregory T

    2005-02-01

    DnaA protein binds bacterial replication origins and it initiates chromosome replication. The Caulobacter crescentus DnaA also initiates chromosome replication and the C. crescentus response regulator CtrA represses chromosome replication. CtrA proteolysis by ClpXP helps restrict chromosome replication to the dividing cell type. We report that C. crescentus DnaA protein is also selectively targeted for proteolysis but DnaA proteolysis uses a different mechanism. DnaA protein is unstable during both growth and stationary phases. During growth phase, DnaA proteolysis ensures that primarily newly made DnaA protein is present at the start of each replication period. Upon entry into stationary phase, DnaA protein is completely removed while CtrA protein is retained. Cell cycle arrest by sudden carbon or nitrogen starvation is sufficient to increase DnaA proteolysis, and relieving starvation rapidly stabilizes DnaA protein. This starvation-induced proteolysis completely removes DnaA protein even while DnaA synthesis continues. Apparently, C. crescentus relies on proteolysis to adjust DnaA in response to such rapid nutritional changes. Depleting the C. crescentus ClpP protease significantly stabilizes DnaA. However, a dominant-negative clpX allele that blocks CtrA degradation, even when combined with a clpA null allele, did not decrease DnaA degradation. We suggest that either a novel chaperone presents DnaA to ClpP or that ClpX is used with exceptional efficiency so that when ClpX activity is limiting for CtrA degradation it is not limiting for DnaA degradation. This unexpected and finely tuned proteolysis system may be an important adaptation for a developmental bacterium that is often challenged by nutrient-poor environments.

  19. Preparation of DNA and nucleoprotein samples for AFM imaging

    PubMed Central

    Lyubchenko, Yuri L.

    2010-01-01

    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. PMID:20864349

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

    PubMed

    Gryson, Nicolas

    2010-03-01

    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.

  1. Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding.

    PubMed Central

    Rinken, R; Thomas, B; Wackernagel, W

    1992-01-01

    Infection of Escherichia coli with phage T4 gene 2am was used to transport 3H-labeled linear duplex DNA into cells to follow its degradation in relation to the cellular genotype. In wild-type cells, 49% of the DNA was made acid soluble within 60 min; in recB or recC cells, only about 5% of the DNA was made acid soluble. Remarkably, in recD cells about 25% of the DNA was rendered acid soluble. The DNA degradation in recD cells depended on intact recB and recC genes. The degradation in recD cells was largely decreased by mutations in recJ (which eliminates the 5' single-strand-specific exonuclease coded by this gene) or xonA (which abolishes the 3' single-strand-specific exonuclease I). In a recD recJ xonA triple mutant, the degradation of linear duplex DNA was roughly at the level of a recB mutant. Results similar to those with the set of recD strains were also obtained with a recC++ mutant (in which the RecD protein is intact but does not function) and its recJ, xonA, and recJ xonA derivatives. The observations provide evidence for a recBC-dependent DNA-unwinding activity that renders unwound DNA susceptible to exonucleolytic degradation. It is proposed that the DNA-unwinding activity causes the efficient recombination, DNA repair, and SOS induction (after application of nalidixic acid) in recD mutants. The RecBC helicase indirectly detected here may have a central function in Chi-dependent recombination and in the recombinational repair of double-strand breaks by the RecBCD pathway. PMID:1322885

  2. Exonuclease TREX1 degrades double-stranded DNA to prevent spontaneous lupus-like inflammatory disease.

    PubMed

    Grieves, Jessica L; Fye, Jason M; Harvey, Scott; Grayson, Jason M; Hollis, Thomas; Perrino, Fred W

    2015-04-21

    The TREX1 gene encodes a potent DNA exonuclease, and mutations in TREX1 cause a spectrum of lupus-like autoimmune diseases. Most lupus patients develop autoantibodies to double-stranded DNA (dsDNA), but the source of DNA antigen is unknown. The TREX1 D18N mutation causes a monogenic, cutaneous form of lupus called familial chilblain lupus, and the TREX1 D18N enzyme exhibits dysfunctional dsDNA-degrading activity, providing a link between dsDNA degradation and nucleic acid-mediated autoimmune disease. We determined the structure of the TREX1 D18N protein in complex with dsDNA, revealing how this exonuclease uses a novel DNA-unwinding mechanism to separate the polynucleotide strands for single-stranded DNA (ssDNA) loading into the active site. The TREX1 D18N dsDNA interactions coupled with catalytic deficiency explain how this mutant nuclease prevents dsDNA degradation. We tested the effects of TREX1 D18N in vivo by replacing the TREX1 WT gene in mice with the TREX1 D18N allele. The TREX1 D18N mice exhibit systemic inflammation, lymphoid hyperplasia, vasculitis, and kidney disease. The observed lupus-like inflammatory disease is associated with immune activation, production of autoantibodies to dsDNA, and deposition of immune complexes in the kidney. Thus, dysfunctional dsDNA degradation by TREX1 D18N induces disease in mice that recapitulates many characteristics of human lupus. Failure to clear DNA has long been linked to lupus in humans, and these data point to dsDNA as a key substrate for TREX1 and a major antigen source in mice with dysfunctional TREX1 enzyme.

  3. Next-Generation Sequencing for Rodent Barcoding: Species Identification from Fresh, Degraded and Environmental Samples

    PubMed Central

    Galan, Maxime; Pagès, Marie; Cosson, Jean-François

    2012-01-01

    Rodentia is the most diverse order among mammals, with more than 2,000 species currently described. Most of the time, species assignation is so difficult based on morphological data solely that identifying rodents at the specific level corresponds to a real challenge. In this study, we compared the applicability of 100 bp mini-barcodes from cytochrome b and cytochrome c oxidase 1 genes to enable rodent species identification. Based on GenBank sequence datasets of 115 rodent species, a 136 bp fragment of cytochrome b was selected as the most discriminatory mini-barcode, and rodent universal primers surrounding this fragment were designed. The efficacy of this new molecular tool was assessed on 946 samples including rodent tissues, feces, museum samples and feces/pellets from predators known to ingest rodents. Utilizing next-generation sequencing technologies able to sequence mixes of DNA, 1,140 amplicons were tagged, multiplexed and sequenced together in one single 454 GS-FLX run. Our method was initially validated on a reference sample set including 265 clearly identified rodent tissues, corresponding to 103 different species. Following validation, 85.6% of 555 rodent samples from Europe, Asia and Africa whose species identity was unknown were able to be identified using the BLASTN program and GenBank reference sequences. In addition, our method proved effective even on degraded rodent DNA samples: 91.8% and 75.9% of samples from feces and museum specimens respectively were correctly identified. Finally, we succeeded in determining the diet of 66.7% of the investigated carnivores from their feces and 81.8% of owls from their pellets. Non-rodent species were also identified, suggesting that our method is sensitive enough to investigate complete predator diets. This study demonstrates how this molecular identification method combined with high-throughput sequencing can open new realms of possibilities in achieving fast, accurate and inexpensive species identification

  4. Next-generation sequencing for rodent barcoding: species identification from fresh, degraded and environmental samples.

    PubMed

    Galan, Maxime; Pagès, Marie; Cosson, Jean-François

    2012-01-01

    Rodentia is the most diverse order among mammals, with more than 2,000 species currently described. Most of the time, species assignation is so difficult based on morphological data solely that identifying rodents at the specific level corresponds to a real challenge. In this study, we compared the applicability of 100 bp mini-barcodes from cytochrome b and cytochrome c oxidase 1 genes to enable rodent species identification. Based on GenBank sequence datasets of 115 rodent species, a 136 bp fragment of cytochrome b was selected as the most discriminatory mini-barcode, and rodent universal primers surrounding this fragment were designed. The efficacy of this new molecular tool was assessed on 946 samples including rodent tissues, feces, museum samples and feces/pellets from predators known to ingest rodents. Utilizing next-generation sequencing technologies able to sequence mixes of DNA, 1,140 amplicons were tagged, multiplexed and sequenced together in one single 454 GS-FLX run. Our method was initially validated on a reference sample set including 265 clearly identified rodent tissues, corresponding to 103 different species. Following validation, 85.6% of 555 rodent samples from Europe, Asia and Africa whose species identity was unknown were able to be identified using the BLASTN program and GenBank reference sequences. In addition, our method proved effective even on degraded rodent DNA samples: 91.8% and 75.9% of samples from feces and museum specimens respectively were correctly identified. Finally, we succeeded in determining the diet of 66.7% of the investigated carnivores from their feces and 81.8% of owls from their pellets. Non-rodent species were also identified, suggesting that our method is sensitive enough to investigate complete predator diets. This study demonstrates how this molecular identification method combined with high-throughput sequencing can open new realms of possibilities in achieving fast, accurate and inexpensive species identification.

  5. Crystal structure of endonuclease G in complex with DNA reveals how it nonspecifically degrades DNA as a homodimer

    PubMed Central

    Lin, Jason L. J.; Wu, Chyuan-Chuan; Yang, Wei-Zen; Yuan, Hanna S.

    2016-01-01

    Endonuclease G (EndoG) is an evolutionarily conserved mitochondrial protein in eukaryotes that digests nucleus chromosomal DNA during apoptosis and paternal mitochondrial DNA during embryogenesis. Under oxidative stress, homodimeric EndoG becomes oxidized and converts to monomers with diminished nuclease activity. However, it remains unclear why EndoG has to function as a homodimer in DNA degradation. Here, we report the crystal structure of the Caenorhabditis elegans EndoG homologue, CPS-6, in complex with single-stranded DNA at a resolution of 2.3 Å. Two separate DNA strands are bound at the ββα-metal motifs in the homodimer with their nucleobases pointing away from the enzyme, explaining why CPS-6 degrades DNA without sequence specificity. Two obligatory monomeric CPS-6 mutants (P207E and K131D/F132N) were constructed, and they degrade DNA with diminished activity due to poorer DNA-binding affinity as compared to wild-type CPS-6. Moreover, the P207E mutant exhibits predominantly 3′-to-5′ exonuclease activity, indicating a possible endonuclease to exonuclease activity change. Thus, the dimer conformation of CPS-6 is essential for maintaining its optimal DNA-binding and endonuclease activity. Compared to other non-specific endonucleases, which are usually monomeric enzymes, EndoG is a unique dimeric endonuclease, whose activity hence can be modulated by oxidation to induce conformational changes. PMID:27738134

  6. Comparison of DNA polymerases for improved forensic analysis of challenging samples.

    PubMed

    Nilsson, Martina; Grånemo, Joakim; Buś, Magdalena M; Havsjö, Mikael; Allen, Marie

    2016-09-01

    Inhibitors of polymerase chain reaction (PCR) amplification often present a challenge in forensic investigations of e.g., terrorism, missing persons, sexual assaults and other criminal cases. Such inhibitors may be counteracted by dilution of the DNA extract, using different additives, and selecting an inhibitory resistant DNA polymerase. Additionally, DNA in forensic samples is often present in limited amounts and degraded, requiring special analyses of short nuclear targets or mitochondrial DNA. The present study evaluated the enzymes AmpliTaq Gold, HotStarTaq Plus, KAPA3G Plant, and KAPA2G Robust, with regard to their ability to overcome inhibitory effects. Our data showed that diluting the extracts and adding bovine serum albumin may increase the yield of the PCR product. However, the largest impact was observed when alternative enzymes were utilized, instead of the commonly used AmpliTaq Gold. KAPA2G Robust presented the highest amplification efficiency in the presence of the inhibitor ammonium nitrate. Moreover, the KAPA3G Plant enzyme had the highest efficiency in amplifying degraded DNA from old buried bone material. KAPA3G Plant and KAPA2G Robust may thus be useful for counteracting inhibitors and improving the analysis of challenging samples.

  7. DNA extraction from aged skeletal samples for STR typing by capillary electrophoresis.

    PubMed

    Huel, René; Amory, Sylvain; Bilić, Ana; Vidović, Stojko; Jasaragić, Edin; Parsons, Thomas J

    2012-01-01

    STR analysis of DNA extracted from skeletal samples can play an important role in the identification of missing persons. Here we present a method for the extraction of DNA from skeletal samples involving complete demineralization and digestion of the sample, followed by purification by silica binding. This method, together with the multiplex STR typing approach also presented, has proven highly successful in the recovery of DNA profiles from degraded, aged skeletal remains from a wide range of environmental contexts. The methodological steps presented include bone decontamination and grinding, DNA extraction, repurification in the case of highly inhibited samples, quantification, STR multiplex amplification, and profile reporting guidelines. However, the conditions applied for amplification and the criteria for allele calling and profile submission must be based on the results of each laboratory's internal validation experiments involving the type of samples relevant to the project at hand. The methods presented here have permitted large-scale DNA-based identification of persons missing from mass disasters and armed conflict.

  8. DNA Degradation in Escherichia coli 15 T-L- Induced by Fast Proton Bombardment

    PubMed Central

    Huston, D. C.; Pollard, E. C.

    1967-01-01

    DNA degradation and its temperature dependence as a function of linear energy transfer were studied in Excherichia coli using fast proton irradiation as the initiating agent. The data indicate that radiation-induced DNA degradation can proceed by two processes. The first, or fast component, begins immediately after irradiation with 60Co γ-rays or with fast protons at doses less than 1010 protons/cm2. The rate is high and involves a maximum of about 50% degradation. It is elicited more efficiently by protons of high linear energy transfer. The second, or slow component, results from higher doses of fast proton bombardment. There is a delay between irradiation and the initiation of this slower component, but 100% of the DNA complement is degraded. The data indicate that both processes are enzyme-mediated, the first probably by normal DNA-related activity and the second by DNAase activity. PMID:4860486

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

    PubMed

    Harteveld, Joyce; Lindenbergh, Alexander; Sijen, Titia

    2013-09-01

    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.

  10. Evaluation of samples comprising minute amounts of DNA.

    PubMed

    Benschop, Corina C G; Haned, Hinda; Yoo, Seong Yeon; Sijen, Titia

    2015-09-01

    Minute amounts of DNA representing only few diploid cells, may be interrogated using enhanced DNA profiling, which will be accompanied by stochastic amplification effects. Notwithstanding, a weight of evidence statistic may be calculated using current interpretation software. In this study, we profiled single donor, two- and three-person samples having only 3 pg to 12 pg of DNA per contributor using both standard and enhanced capillary electrophoresis (CE) injection settings. Likelihood ratios (LRs) were computed using LRmix Studio, compared for both types of profiles and examined in relation to the amount of DNA, drop-out level, number of detected alleles, peak heights and reproducibility of alleles. Especially for DNA profiles that were generated using enhanced CE, the obtained LRs could indicate strong evidence in favour of the prosecution (log10(LR)>6), also when the amount of DNA represented about half of a diploid cell equivalent in the amplification. These results illustrate that an assessment of the criminalistic relevance of a sample carrying minute amounts of DNA is essential prior to applying enhanced interrogation techniques and/or calculating a weight of evidence statistic.

  11. Microfluidic devices for DNA sequencing: sample preparation and electrophoretic analysis.

    PubMed

    Paegel, Brian M; Blazej, Robert G; Mathies, Richard A

    2003-02-01

    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 time and reagent consumption, and eliminates costly and unreliable macroscale robotics and laboratory apparatus. A microfabricated device for high-throughput DNA sequencing that couples clone isolation, template amplification, Sanger extension, purification, and electrophoretic analysis in a single microfluidic circuit is now attainable.

  12. Comparison of three methods of DNA extraction from human bones with different degrees of degradation.

    PubMed

    Jakubowska, Joanna; Maciejewska, Agnieszka; Pawłowski, Ryszard

    2012-01-01

    There is a necessity for deceased identification as a result of many accidents and sometimes bones are the only accessible source of DNA. So far, a universal method that allows for extraction of DNA from materials at different stages of degradation does not exist. The aims of this study were: the comparison of three methods of DNA extraction from bones with different degree of degradation and an evaluation of the usefulness of these methods in forensic genetics. The efficiency of DNA extraction, the degree of extract contamination by polymerase chain reaction (PCR) inhibitors and the possibility of determining the STR loci profile were especially being compared. Nuclear DNA from bones at different states of degradation was isolated using three methods: classical, organic phenol-chloroform extraction, DNA extraction from crystal aggregates and extraction by total demineralisation. Total demineralisation is the best method for most cases of DNA extraction from bones, although it does not provide pure DNA. DNA extraction from aggregates removes inhibitors much better and is also a good method of choice when identity determination of exhumed remains is necessary. In the case of not buried bones (remains found outside) total demineralisation or phenol-chloroform protocols are more efficient for successful DNA extraction.

  13. Exonuclease VII is involved in "reckless" DNA degradation in UV-irradiated Escherichia coli.

    PubMed

    Repar, Jelena; Briški, Nina; Buljubašić, Maja; Zahradka, Ksenija; Zahradka, Davor

    2013-01-20

    The recA mutants of Escherichia coli exhibit an abnormal DNA degradation that starts at sites of double-strand DNA breaks (DSBs), and is mediated by RecBCD exonuclease (ExoV). This "reckless" DNA degradation occurs spontaneously in exponentially growing recA cells, and is stimulated by DNA-damaging agents. We have previously found that the xonA and sbcD mutations, which inactivate exonuclease I (ExoI) and SbcCD nuclease, respectively, markedly suppress "reckless" DNA degradation in UV-irradiated recA cells. In the present work, we show that inactivation of exonuclease VII (ExoVII) by an xseA mutation contributes to attenuation of DNA degradation in UV-irradiated recA mutants. The xseA mutation itself has only a weak effect, however, it acts synergistically with the xonA or sbcD mutations in suppressing "reckless" DNA degradation. The quadruple xseA xonA sbcD recA mutants show no sign of DNA degradation during post-irradiation incubation, suggesting that ExoVII, together with ExoI and SbcCD, plays a crucial role in regulating RecBCD-catalyzed chromosome degradation. We propose that these nucleases act on DSBs to create blunt DNA ends, the preferred substrates for the RecBCD enzyme. In addition, our results show that in UV-irradiated recF recA(+) cells, the xseA, xonA, and sbcD mutations do not affect RecBCD-mediated DNA repair, suggesting that ExoVII, ExoI and SbcCD nucleases are not essential for the initial targeting of RecBCD to DSBs. It is possible that the DNA-blunting activity provided by ExoVII, ExoI and SbcCD is required for an exchange of RecBCD molecules on dsDNA ends during ongoing "reckless" DNA degradation.

  14. Recent advances and considerations for the future in forensic analysis of degraded DNA by autosomic miniSTR multiplex genotyping.

    PubMed

    Odriozola, A; Aznar, J M; Celorrio, D; De Pancorbo, M M

    2011-08-01

    STR genotyping from degraded DNA samples requires genetic profiles to be obtained from DNA fragments no bigger than 200-300 bp. It requires the use of miniSTRs, which are smaller than the STRs used in standard typing. This paper reviews recent advances in miniSTR genotyping, beginning with a brief introduction to the processes involved in DNA fragmentation and how it hinders standard STR genotyping before proceeding further to the loci included in the main DNA databases and finishing with the International Workgroups' recommended design strategies for developing miniSTR reactions. The results of the efforts of many laboratories achieving different STR multiplexes and patents are also described and compared. Finally, a consideration of the perspectives for the future in this area is presented.

  15. Ancient DNA studies: new perspectives on old samples

    PubMed Central

    2012-01-01

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

  16. Residual soil DNA extraction increases the discriminatory power between samples.

    PubMed

    Young, Jennifer M; Weyrich, Laura S; Clarke, Laurence J; Cooper, Alan

    2015-06-01

    Forensic soil analysis relies on capturing an accurate and reproducible representation of the diversity from limited quantities of soil; however, inefficient DNA extraction can markedly alter the taxonomic abundance. The performance of a standard commercial DNA extraction kit (MOBIO PowerSoil DNA Isolation kit) and three modified protocols of this kit: soil pellet re-extraction (RE); an additional 24-h lysis incubation step at room temperature (RT); and 24-h lysis incubation step at 55°C (55) were compared using high-throughput sequencing of the internal transcribed spacer I ribosomal DNA. DNA yield was not correlated with fungal diversity and the four DNA extraction methods displayed distinct fungal community profiles for individual samples, with some phyla detected exclusively using the modified methods. Application of a 24 h lysis step will provide a more complete inventory of fungal biodiversity, and re-extraction of the residual soil pellet offers a novel tool for increasing discriminatory power between forensic soil samples.

  17. Preamplification Procedure for the Analysis of Ancient DNA Samples

    PubMed Central

    Del Gaudio, Stefania; Cirillo, Alessandra; Di Bernardo, Giovanni; Galderisi, Umberto; Thanassoulas, Theodoros; Pitsios, Theodoros; Cipollaro, Marilena

    2013-01-01

    In ancient DNA studies the low amount of endogenous DNA represents a limiting factor that often hampers the result achievement. In this study we extracted the DNA from nine human skeletal remains of different ages found in the Byzantine cemetery of Abdera Halkidiki and in the medieval cemetery of St. Spiridion in Rhodes (Greece). Real-time quantitative polymerase chain reaction (qPCR) was used to detect in the extracts the presence of PCR inhibitors and to estimate the DNA content. As mitochondrial DNA was detected in all samples, amplification of nuclear targets, as amelogenin and the polymorphism M470V of the transmembrane conductance regulator gene, yielded positive results in one case only. In an effort to improve amplification success, we applied, for the first time in ancient DNA, a preamplification strategy based on TaqMan PreAmp Master Mix. A comparison between results obtained from nonpreamplified and preamplified samples is reported. Our data, even if preliminary, show that the TaqMan PreAmp procedure may improve the sensitivity of qPCR analysis. PMID:24187523

  18. Preamplification procedure for the analysis of ancient DNA samples.

    PubMed

    Del Gaudio, Stefania; Cirillo, Alessandra; Di Bernardo, Giovanni; Galderisi, Umberto; Thanassoulas, Theodoros; Pitsios, Theodoros; Cipollaro, Marilena

    2013-01-01

    In ancient DNA studies the low amount of endogenous DNA represents a limiting factor that often hampers the result achievement. In this study we extracted the DNA from nine human skeletal remains of different ages found in the Byzantine cemetery of Abdera Halkidiki and in the medieval cemetery of St. Spiridion in Rhodes (Greece). Real-time quantitative polymerase chain reaction (qPCR) was used to detect in the extracts the presence of PCR inhibitors and to estimate the DNA content. As mitochondrial DNA was detected in all samples, amplification of nuclear targets, as amelogenin and the polymorphism M470V of the transmembrane conductance regulator gene, yielded positive results in one case only. In an effort to improve amplification success, we applied, for the first time in ancient DNA, a preamplification strategy based on TaqMan PreAmp Master Mix. A comparison between results obtained from nonpreamplified and preamplified samples is reported. Our data, even if preliminary, show that the TaqMan PreAmp procedure may improve the sensitivity of qPCR analysis.

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

    PubMed

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

    2011-12-01

    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 DNA by alkaline lysis method were higher than by CTAB (cetyltrimethylammonium bromide) method. For sweet rice wine, CAMV35S (cauliflower mosaic virus 35S) promoter and NOS (nopaline synthase) terminator were degraded by the third day, whereas the exogenous gene Bar (bialaphos resistance) remained unaffected. For rice crackers, boiling, drying and microwaving contributed to the initial degradations of DNA. Baking resulted in further degradations, and frying led to the most severe changes. These results indicated that the stability of DNA in GM rice was different under different processing conditions. For sweet rice wine, Bar was most stable, followed by NOS, CAMV35S, and SPS. For rice crackers, CAMV35S was most stable, followed by SPS, NOS, and Bar.

  20. Action of hydrogen peroxide on degradation of DNA after irradiation in Escherichia coli.

    PubMed

    Keller, K M; Pollard, E C

    1977-05-01

    Hydrogen peroxide (H2O2), which produces breaks in cellular DNA, has not hitherto been shown to cause degradation of DNA. In this investigation it is shown that if transcription is blocked with rifampin, treatment with H2O2 causes degradation of DNA to nearly the same extent as does gamma-radiation. Further, if cells are given a treatment with H2O2 and incubated for 50 min, the amount of degradation in a second treatment is markedly less. This is attributed to the induction of the inhibitor of post-irradiation degradation of DNA (prd) by the first treatment. There is thus a double action of H2O2: first, to induce inhibition, and second, to cause degradation of DNA to begin in non-induced cells. The genetic dependence of induction by H2O2 mimics that of ionizing radiation. Accordingly, the induction process does not occur in recA- and lex- cells, because they are not inducible and is absent in recB- cells because they lack exonuclease V, the major component of prd. Potassium iodide (KI), an OH radical scavenger, negates the action of peroxide on DNA. The results obtained in this study suggest a possible theory for the evolution of radiation response systems

  1. Assessing PreCR™ repair enzymes for restoration of STR profiles from artificially degraded DNA for human identification.

    PubMed

    Robertson, James M; Dineen, Shauna M; Scott, Kristina A; Lucyshyn, Jonathan; Saeed, Maria; Murphy, Devonie L; Schweighardt, Andrew J; Meiklejohn, Kelly A

    2014-09-01

    Forensic scientists have used several approaches to obtain short tandem repeat (STR) profiles from compromised DNA samples, including supplementing the polymerase chain reaction (PCR) with enhancers and using procedures yielding reduced-length amplicons. For degraded DNA, the peak intensities of the alleles separated by electrophoresis generally decrease as the length of the allele increases. When the intensities of the alleles decrease below an established threshold, they are described as drop-outs, thus contributing to a partial STR profile. This work assesses the use of repair enzymes to improve the STR profiles from artificially degraded DNA. The commercial PreCR™ repair kit of DNA repair enzymes was tested on both purified DNA and native DNA in body fluids exposed to oxidizing agents, hydrolytic conditions, ultraviolet (UV) and ionizing radiation, and desiccation. The strategy was to restrict the level of DNA damage to that which yields partial STR profiles in order to test for allele restoration as opposed to simple allele enhancement. Two protocols were investigated for allele restoration: a sequential protocol using the manufacturer's repair procedure and a modified protocol reportedly designed for optimal STR analysis of forensic samples. Allele restoration was obtained with both protocols, but the peak height appeared to be higher for the modified protocol (determined by Mann-Kendall Trend Test). The success of the approach using the PreCR™ repair enzymes was sporadic; it led to allele restoration as well as allele drop-out. Additionally, allele restoration with the PreCR™ enzymes was compared with restoration by alternative, but commonly implemented approaches using Restorase™, PCRBoost™, bovine serum albumin (BSA) and the Minifiler™ STR system. The alternative methods were also successful in improving the STR profile, but their success also depended on the quality of the template encountered. Our results indicate the PreCR™ repair kit may

  2. Maternal inheritance of mitochondrial DNA: degradation of paternal mitochondria by allogeneic organelle autophagy, allophagy.

    PubMed

    Sato, Miyuki; Sato, Ken

    2012-03-01

    Maternal inheritance of mitochondrial DNA (mtDNA) is generally observed in many eukaryotes. Sperm-derived paternal mitochondria and their mtDNA enter the oocyte cytoplasm upon fertilization and then normally disappear during early embryogenesis. However, the mechanism underlying this clearance of paternal mitochondria has remained largely unknown. Recently, we showed that autophagy is required for the elimination of paternal mitochondria in Caenorhabditis elegans embryos. Shortly after fertilization, autophagosomes are induced locally around the penetrated sperm components. These autophagosomes engulf paternal mitochondria, resulting in their lysosomal degradation during early embryogenesis. In autophagy-defective zygotes, paternal mitochondria and their genomes remain even in the larval stage. Therefore, maternal inheritance of mtDNA is accomplished by autophagic degradation of paternal mitochondria. We also found that another kind of sperm-derived structure, called the membranous organelle, is degraded by zygotic autophagy as well. We thus propose to term this allogeneic (nonself) organelle autophagy as allophagy.

  3. Searching for the Optimal Sampling Solution: Variation in Invertebrate Communities, Sample Condition and DNA Quality

    PubMed Central

    Gossner, Martin M.; Struwe, Jan-Frederic; Sturm, Sarah; Max, Simeon; McCutcheon, Michelle; Weisser, Wolfgang W.; Zytynska, Sharon E.

    2016-01-01

    There is a great demand for standardising biodiversity assessments in order to allow optimal comparison across research groups. For invertebrates, pitfall or flight-interception traps are commonly used, but sampling solution differs widely between studies, which could influence the communities collected and affect sample processing (morphological or genetic). We assessed arthropod communities with flight-interception traps using three commonly used sampling solutions across two forest types and two vertical strata. We first considered the effect of sampling solution and its interaction with forest type, vertical stratum, and position of sampling jar at the trap on sample condition and community composition. We found that samples collected in copper sulphate were more mouldy and fragmented relative to other solutions which might impair morphological identification, but condition depended on forest type, trap type and the position of the jar. Community composition, based on order-level identification, did not differ across sampling solutions and only varied with forest type and vertical stratum. Species richness and species-level community composition, however, differed greatly among sampling solutions. Renner solution was highly attractant for beetles and repellent for true bugs. Secondly, we tested whether sampling solution affects subsequent molecular analyses and found that DNA barcoding success was species-specific. Samples from copper sulphate produced the fewest successful DNA sequences for genetic identification, and since DNA yield or quality was not particularly reduced in these samples additional interactions between the solution and DNA must also be occurring. Our results show that the choice of sampling solution should be an important consideration in biodiversity studies. Due to the potential bias towards or against certain species by Ethanol-containing sampling solution we suggest ethylene glycol as a suitable sampling solution when genetic analysis

  4. Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation.

    PubMed

    Vallerga, María Belén; Mansilla, Sabrina F; Federico, María Belén; Bertolin, Agustina P; Gottifredi, Vanesa

    2015-12-01

    After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates.

  5. Rad51 recombinase prevents Mre11 nuclease-dependent degradation and excessive PrimPol-mediated elongation of nascent DNA after UV irradiation

    PubMed Central

    Vallerga, María Belén; Mansilla, Sabrina F.; Federico, María Belén; Bertolin, Agustina P.; Gottifredi, Vanesa

    2015-01-01

    After UV irradiation, DNA polymerases specialized in translesion DNA synthesis (TLS) aid DNA replication. However, it is unclear whether other mechanisms also facilitate the elongation of UV-damaged DNA. We wondered if Rad51 recombinase (Rad51), a factor that escorts replication forks, aids replication across UV lesions. We found that depletion of Rad51 impairs S-phase progression and increases cell death after UV irradiation. Interestingly, Rad51 and the TLS polymerase polη modulate the elongation of nascent DNA in different ways, suggesting that DNA elongation after UV irradiation does not exclusively rely on TLS events. In particular, Rad51 protects the DNA synthesized immediately before UV irradiation from degradation and avoids excessive elongation of nascent DNA after UV irradiation. In Rad51-depleted samples, the degradation of DNA was limited to the first minutes after UV irradiation and required the exonuclease activity of the double strand break repair nuclease (Mre11). The persistent dysregulation of nascent DNA elongation after Rad51 knockdown required Mre11, but not its exonuclease activity, and PrimPol, a DNA polymerase with primase activity. By showing a crucial contribution of Rad51 to the synthesis of nascent DNA, our results reveal an unanticipated complexity in the regulation of DNA elongation across UV-damaged templates. PMID:26627254

  6. Filtration recovery of extracellular DNA from environmental water samples

    EPA Science Inventory

    qPCR methods are able to analyze DNA from microbes within hours of collecting water samples, providing the promptest notification and public awareness possible when unsafe pathogenic levels are reached. Health risk, however, may be overestimated by the presence of extracellular ...

  7. Microbial diversity in degraded and non-degraded petroleum samples and comparison across oil reservoirs at local and global scales.

    PubMed

    Sierra-Garcia, Isabel Natalia; Dellagnezze, Bruna M; Santos, Viviane P; Chaves B, Michel R; Capilla, Ramsés; Santos Neto, Eugenio V; Gray, Neil; Oliveira, Valeria M

    2017-01-01

    Microorganisms have shown their ability to colonize extreme environments including deep subsurface petroleum reservoirs. Physicochemical parameters may vary greatly among petroleum reservoirs worldwide and so do the microbial communities inhabiting these different environments. The present work aimed at the characterization of the microbiota in biodegraded and non-degraded petroleum samples from three Brazilian reservoirs and the comparison of microbial community diversity across oil reservoirs at local and global scales using 16S rRNA clone libraries. The analysis of 620 16S rRNA bacterial and archaeal sequences obtained from Brazilian oil samples revealed 42 bacterial OTUs and 21 archaeal OTUs. The bacterial community from the degraded oil was more diverse than the non-degraded samples. Non-degraded oil samples were overwhelmingly dominated by gammaproteobacterial sequences with a predominance of the genera Marinobacter and Marinobacterium. Comparisons of microbial diversity among oil reservoirs worldwide suggested an apparent correlation of prokaryotic communities with reservoir temperature and depth and no influence of geographic distance among reservoirs. The detailed analysis of the phylogenetic diversity across reservoirs allowed us to define a core microbiome encompassing three bacterial classes (Gammaproteobacteria, Clostridia, and Bacteroidia) and one archaeal class (Methanomicrobia) ubiquitous in petroleum reservoirs and presumably owning the abilities to sustain life in these environments.

  8. Use of quantitative real-time PCR to estimate maize endogenous DNA degradation after cooking and extrusion or in food products.

    PubMed

    Murray, Sarah R; Butler, Ruth C; Hardacre, Allan K; Timmerman-Vaughan, Gail M

    2007-03-21

    Polymerase chain reaction (PCR) is being used increasingly to detect DNA sequences for food quality testing for GM content, microbial contamination, and ingredient content. However, food processing often results in DNA degradation and therefore may affect the suitability of PCR or even DNA sequence detection for food quality assurance. This paper describes a novel approach using quantitative real-time PCR (qPCR) to estimate the extent of DNA degradation. With use of two maize endogenous nuclear sequences, sets of four qPCR assays were developed to amplify target sequences ranging from<100 bp to approximately 1000 bp. The maize nuclear sequences used encode chloroplastic glyceraldehyde-3-phosphate dehydrogenase and cell wall invertase. The utility of the qPCR approach for quantifying the effective concentration of maize DNA that is needed to amplify variable length DNA sequences was demonstrated using samples of maize cornmeal cooked in water for variable times, extrusion products developed using different barrel temperature and torque settings, and a range of food products from supermarket shelves. Results showed that maize DNA was substantially degraded by a number of processing procedures, including cooking for 5 min or more, extrusion at high temperatures and/or high torque settings, and in most processed foods from supermarket shelves. Processing also reduced the effective concentration of DNA sequences capable of directing amplification of the <100 bp assays as well, particularly after popping of popping corn or extrusion at a combination of high temperature and torque settings. The approach for quantifying DNA degradation described in this paper may also be of use in disciplines where understanding the extent of DNA degradation is important, such as in environmental, forensic, or historical samples.

  9. Bacterial and fungal DNA extraction from blood samples: manual protocols.

    PubMed

    Lorenz, Michael G; Mühl, Helge; Disqué, Claudia

    2015-01-01

    A critical point of molecular diagnosis of systemic infections is the method employed for the extraction of microbial DNA from blood. A DNA isolation method has to be able to fulfill several fundamental requirements for optimal performance of diagnostic assays. First of all, low- and high-molecular-weight substances of the blood inhibitory to downstream analytical reactions like PCR amplification have to be removed. This includes human DNA which is a known source of false-positive results and factor decreasing the analytical sensitivity of PCR assays by unspecific primer binding. At the same time, even extremely low amounts of microbial DNA need to be supplied to molecular diagnostic assays in order to detect low pathogen loads in the blood. Further, considering the variety of microbial etiologies of sepsis, a method should be capable of lysing Gram-positive, Gram-negative, and fungal organisms. Last, extraction buffers, reagents, and consumables have to be free of microbial DNA which leads to false-positive results. Here, we describe manual methods which allow the extraction of microbial DNA from small- and large-volume blood samples for the direct molecular analysis of pathogen.

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

    PubMed

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

    2013-08-15

    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.

  11. Bioaugmentation of bromoamine acid degradation with Sphingomonas xenophaga QYY and DNA fingerprint analysis of augmented systems.

    PubMed

    Qu, Yuanyuan; Zhou, Jiti; Wang, Jing; Song, Zhiyong; Xing, Linlin; Fu, Xiang

    2006-02-01

    One high-effective bromoamine acid (1-amino-4-bromoanthraquinone-2-sulfonic acid, BAA) degrading strain was isolated previously with the ability to use BAA as sole source of carbon and nitrogen. It was identified as Sphingomonas xenophaga QYY by 16S rDNA sequence analysis and physio-biochemical tests. In this study, bioaugmentation of BAA degradation with suspended and immobilized cells of strain QYY was investigated. The optimal degradation conditions were as follows: temperature 30 degrees C, pH 6.0-7.0, 150 rev min(-1) and the immobilized cells maintained degradation activity to BAA after 60 days storage at 4 degrees C. The structure of BAA was evidently changed according to the analysis of total organic carbon removal of BAA (about 50%) and the UV-VIS spectra changes during the biodegradation. Bioaugmented systems exhibited stronger abilities degrading BAA than the non-bioaugmented control ones. And microbial community dynamics of augmented systems was revealed by amplified ribosomal DNA restriction analysis (ARDRA), a modern DNA fingerprint technique. The results indicated that the microbial community dynamics was substantially changed throughout the augmentation process. This study suggests that it is feasible and potentially useful to enhance BAA degradation using bioaugmentation with the immobilized cells of BAA-degrading bacterium.

  12. Environmental DNA (eDNA) sampling improves occurrence and detection estimates of invasive Burmese pythons

    USGS Publications Warehouse

    Hunter, Margaret E.; Oyler-McCance, Sara J.; Dorazio, Robert M.; Fike, Jennifer A.; Smith, Brian J.; Hunter, Charles T.; Reed, Robert N.; Hart, Kristen M.

    2015-01-01

    Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors

  13. Environmental DNA (eDNA) Sampling Improves Occurrence and Detection Estimates of Invasive Burmese Pythons

    PubMed Central

    Hunter, Margaret E.; Oyler-McCance, Sara J.; Dorazio, Robert M.; Fike, Jennifer A.; Smith, Brian J.; Hunter, Charles T.; Reed, Robert N.; Hart, Kristen M.

    2015-01-01

    Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors

  14. Genotyping of plant and animal samples without prior DNA purification.

    PubMed

    Chum, Pak Y; Haimes, Josh D; André, Chas P; Kuusisto, Pia K; Kelley, Melissa L

    2012-09-24

    The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors. PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS) is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA

  15. Hydrodynamic effects in buccal cell DNA sample collection

    NASA Astrophysics Data System (ADS)

    Aidun, C. K.; Sozer, A. C.

    2003-11-01

    Many different methods can be used for collection of biological samples from individuals for DNA profiling purposes. However, blood and buccal cells are the two most popular sources of DNA. In situations where large numbers of samples have to be collected, buccal cell collection methods are the preferred choice because of minimized health risks and ease of collection, transportation, and storage. The normal practice in buccal cell collection is to rub a cotton swab or a piece of paper through the inner check of the subject in order to release the cells and to attach and collect the cells on the cotton or paper fibers. The problem with the current forms of sample collection and storage is that in some cases up to 20% of the samples collected do not result in a DNA profile of adequate quality to be reported by the laboratory without repeated testing. In this study, we consider the mechanics of a small sheet of paper being rubbed on the surface of the inner check. The process is not too different from coating a paper substrate with highly deformable material. The shear field developed between the paper and the fluid adjacent to the cells are estimated based on the available data. The action of the cell release and the cell adherence to the surface will be outlined.

  16. DNA degradation and genetic analysis of empty puparia: genetic identification limits in forensic entomology.

    PubMed

    Mazzanti, Morena; Alessandrini, Federica; Tagliabracci, Adriano; Wells, Jeffrey D; Campobasso, Carlo P

    2010-02-25

    Puparial cases are common remnants of necrophagous flies in crime investigations. They usually represent the longest developmental time and, therefore, they can be very useful for the estimation of the post-mortem interval (PMI). However, before any PMI estimate, it is crucial to identify the species of fly eclosed from each puparium associated with the corpse. Morphological characteristics of the puparium are often distinctive enough to permit a species identification. But, even an accurate morphological analysis of empty puparia cannot discriminate among different species of closely related flies. Furthermore, morphological identification may be impossible if the fly puparia are poorly preserved or in fragments. This study explores the applicability of biomolecular techniques on empty puparia and their fragments for identification purposes. A total of 63 empty puparia of necrophagous Diptera resulting from forensic casework were examined. Samples were divided into three groups according to size, type and time of eclosion in order to verify whether the physical characteristics and puparia weathering can influence the amount of DNA extraction. The results suggest that a reliable genetic identification of forensically important flies may also be performed from empty puparia and/or their fragments. However, DNA degradation can deeply compromise the genetic analysis since the older the fly puparia, the smaller are the amplified fragments.

  17. Exploring effective sampling design for monitoring soil organic carbon in degraded Tibetan grasslands.

    PubMed

    Chang, Xiaofeng; Bao, Xiaoying; Wang, Shiping; Zhu, Xiaoxue; Luo, Caiyun; Zhang, Zhenhua; Wilkes, Andreas

    2016-05-15

    The effects of climate change and human activities on grassland degradation and soil carbon stocks have become a focus of both research and policy. However, lack of research on appropriate sampling design prevents accurate assessment of soil carbon stocks and stock changes at community and regional scales. Here, we conducted an intensive survey with 1196 sampling sites over an area of 190 km(2) of degraded alpine meadow. Compared to lightly degraded meadow, soil organic carbon (SOC) stocks in moderately, heavily and extremely degraded meadow were reduced by 11.0%, 13.5% and 17.9%, respectively. Our field survey sampling design was overly intensive to estimate SOC status with a tolerable uncertainty of 10%. Power analysis showed that the optimal sampling density to achieve the desired accuracy would be 2, 3, 5 and 7 sites per 10 km(2) for lightly, moderately, heavily and extremely degraded meadows, respectively. If a subsequent paired sampling design with the optimum sample size were performed, assuming stock change rates predicted by experimental and modeling results, we estimate that about 5-10 years would be necessary to detect expected trends in SOC in the top 20 cm soil layer. Our results highlight the utility of conducting preliminary surveys to estimate the appropriate sampling density and avoid wasting resources due to over-sampling, and to estimate the sampling interval required to detect an expected sequestration rate. Future studies will be needed to evaluate spatial and temporal patterns of SOC variability.

  18. DNA methylome profiling using neonatal dried blood spot samples: a proof-of-principle study.

    PubMed

    Hollegaard, Mads Vilhelm; Grauholm, Jonas; Nørgaard-Pedersen, Bent; Hougaard, David Michael

    2013-04-01

    spot references. This indicates that two 3.2mm disks contain enough material for reliable methylome profiling and that storing the whole-blood sample on neonatal dried blood spot filter paper for 3years does not interfere with the outcome of the analysis. Furthermore, we compared the adult DNA methylation profile to the neonatal dried blood spot sample profile. Approximately 50 sites in the subjects were significantly (P<0.001) different in the newborn sample compared with the adult sample. Both being healthy adults and the high quality of the DNA methylation array led to the conclusion that the archived neonatal dried blood spot samples can be used for methylome profiling, despite decades of storage and DNA degradation. In conclusion, we show that reliable methylome data can be obtained from old neonatal dried blood spot samples, by using a reasonable amount of the limited resource. This further adds to the use of neonatal dried blood spot samples in genetic research and screening and paves the way for unique population-based studies of epigenetic modifications after birth.

  19. Application of environmental DNA analysis for the detection of Opisthorchis viverrini DNA in water samples.

    PubMed

    Hashizume, Hiroki; Sato, Megumi; Sato, Marcello Otake; Ikeda, Sumire; Yoonuan, Tippayarat; Sanguankiat, Surapol; Pongvongsa, Tiengkham; Moji, Kazuhiko; Minamoto, Toshifumi

    2017-05-01

    Opisthorchiasis, which can lead to cholangiocarcinoma in cases of chronic infection, is a major public health problem in Southeast Asian countries. The trematode, Opisthorchis viverrini, is the causative agent of the disease. Accurate and rapid monitoring of O. viverrini is crucial for disease prevention and containment. Therefore, in this study we sought to develop a novel species-specific real-time PCR assay for detecting O. viverrini using environmental DNA (eDNA). The diagnostic sensitivity of the newly developed real-time PCR assay was similar to that of the traditional PCR assay for 50 fecal samples collected in Lao PDR (21 and 19 samples were positive by real-time PCR and traditional PCR, respectively). The efficacy of eDNA analysis and its applicability in the field were tested using a total of 94 environmental water samples collected from 44 sites in Savannakhet, Lao PDR during May and October 2015 and February 2016. O. viverrini eDNA was detected in five samples by real-time PCR, indicating the presence of the fluke in the area and the risk of infection for individuals consuming fish from these water sources. The application of eDNA analysis would facilitate the identification of O. viverrini endemic hotspots and contribute to the ecological control of opisthorchiasis, and this strategy can be applied to other eukaryotic water pathogens.

  20. 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

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

    2002-01-01

    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. PMID:12088997

  1. Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

    NASA Technical Reports Server (NTRS)

    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

    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.

  2. Environmental DNA sampling protocol - filtering water to capture DNA from aquatic organisms

    USGS Publications Warehouse

    Laramie, Matthew B.; Pilliod, David S.; Goldberg, Caren S.; Strickler, Katherine M.

    2015-09-29

    Environmental DNA (eDNA) analysis is an effective method of determining the presence of aquatic organisms such as fish, amphibians, and other taxa. This publication is meant to guide researchers and managers in the collection, concentration, and preservation of eDNA samples from lentic and lotic systems. A sampling workflow diagram and three sampling protocols are included as well as a list of suggested supplies. Protocols include filter and pump assembly using: (1) a hand-driven vacuum pump, ideal for sample collection in remote sampling locations where no electricity is available and when equipment weight is a primary concern; (2) a peristaltic pump powered by a rechargeable battery-operated driver/drill, suitable for remote sampling locations when weight consideration is less of a concern; (3) a 120-volt alternating current (AC) powered peristaltic pump suitable for any location where 120-volt AC power is accessible, or for roadside sampling locations. Images and detailed descriptions are provided for each step in the sampling and preservation process.

  3. ATM mediates repression of DNA end-degradation in an ATP-dependent manner.

    PubMed

    Rahal, Elias A; Henricksen, Leigh A; Li, Yuling; Turchi, John J; Pawelczak, Katherine S; Dixon, Kathleen

    2008-03-01

    Ataxia telangiectasia mutated (ATM) is a PI3-kinase-like kinase (PIKK) associated with DNA double-strand break (DSB) repair and cell cycle control. We have previously reported comparable efficiencies of DSB repair in nuclear extracts from both ATM deficient (A-T) and control (ATM+) cells; however, the repair products from the A-T nuclear extracts contained deletions encompassing longer stretches of DNA compared to controls. These deletions appeared to result from end-joining at sites of microhomology. These data suggest that ATM hinders error-prone repair pathways that depend on degradation of DNA ends at a break. Such degradation may account for the longer deletions we formerly observed in A-T cell extracts. To address this possibility we assessed the degradation of DNA duplex substrates in A-T and control nuclear extracts under DSB repair conditions. We observed a marked shift in signal intensity from full-length products to shorter products in A-T nuclear extracts, and addition of purified ATM to A-T nuclear extracts restored full-length product detection. This repression of degradation by ATM was both ATP-dependent and inhibited by the PIKK inhibitors wortmannin and caffeine. Addition of pre-phosphorylated ATM to an A-T nuclear extract in the presence of PIKK inhibitors was insufficient in repressing degradation, indicating that kinase activities are required. These results demonstrate a role for ATM in preventing the degradation of DNA ends possibly through repressing nucleases implicated in microhomology-mediated end-joining.

  4. Bacterial Argonaute samples the transcriptome to identify foreign DNA

    PubMed Central

    Olovnikov, Ivan; Chan, Ken; Sachidanandam, Ravi; Newman, Dianne K.; Aravin, Alexei A.

    2013-01-01

    summary Eukaryotic Argonautes bind small RNAs and use them as guides to find complementary RNA targets and induce gene silencing. Though homologs of eukaryotic Argonautes are present in many bacteria and archaea their small RNA partners and functions are unknown. We found that the Argonaute of Rhodobacter sphaeroides (RsAgo) associates with 15-19 nt RNAs that correspond to the majority of transcripts. RsAgo also binds single-stranded 22-24 nt DNA molecules that are complementary to the small RNAs and enriched in sequences derived from exogenous plasmids as well as genome-encoded foreign nucleic acids such as transposons and phage genes. Expression of RsAgo in the heterologous E. coli system leads to formation of plasmid– derived small RNA and DNA and plasmid degradation. In a R. sphaeroides mutant lacking RsAgo, expression of plasmid-encoded genes is elevated. Our results indicate that RNAi-related processes found in eukaryotes are also conserved in bacteria and target foreign nucleic acids. PMID:24034694

  5. Developmental validation of the PrepFiler Forensic DNA Extraction Kit for extraction of genomic DNA from biological samples.

    PubMed

    Brevnov, Maxim G; Pawar, Hemant S; Mundt, Janna; Calandro, Lisa M; Furtado, Manohar R; Shewale, Jaiprakash G

    2009-05-01

    The PrepFiler Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 microL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA paper), and touch evidence-type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol-chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.

  6. Soil sampling and isolation of extracellular DNA from large amount of starting material suitable for metabarcoding studies.

    PubMed

    Taberlet, Pierre; Prud'Homme, Sophie M; Campione, Etienne; Roy, Julien; Miquel, Christian; Shehzad, Wasim; Gielly, Ludovic; Rioux, Delphine; Choler, Philippe; Clément, Jean-Christophe; Melodelima, Christelle; Pompanon, François; Coissac, Eric

    2012-04-01

    DNA metabarcoding refers to the DNA-based identification of multiple species from a single complex and degraded environmental sample. We developed new sampling and extraction protocols suitable for DNA metabarcoding analyses targeting soil extracellular DNA. The proposed sampling protocol has been designed to reduce, as much as possible, the influence of local heterogeneity by processing a large amount of soil resulting from the mixing of many different cores. The DNA extraction is based on the use of saturated phosphate buffer. The sampling and extraction protocols were validated first by analysing plant DNA from a set of 12 plots corresponding to four plant communities in alpine meadows, and, second, by conducting pilot experiments on fungi and earthworms. The results of the validation experiments clearly demonstrated that sound biological information can be retrieved when following these sampling and extraction procedures. Such a protocol can be implemented at any time of the year without any preliminary knowledge of specific types of organisms during the sampling. It offers the opportunity to analyse all groups of organisms using a single sampling/extraction procedure and opens the possibility to fully standardize biodiversity surveys.

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

    PubMed

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

    2000-11-01

    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.

  8. 28 CFR 28.13 - Analysis and indexing of DNA samples.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 1 2010-07-01 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 § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau...

  9. 28 CFR 28.13 - Analysis and indexing of DNA samples.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 28 Judicial Administration 1 2014-07-01 2014-07-01 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 § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau...

  10. 28 CFR 28.13 - Analysis and indexing of DNA samples.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 1 2011-07-01 2011-07-01 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 § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau...

  11. 28 CFR 28.13 - Analysis and indexing of DNA samples.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 28 Judicial Administration 1 2012-07-01 2012-07-01 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 § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau...

  12. 28 CFR 28.13 - Analysis and indexing of DNA samples.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 28 Judicial Administration 1 2013-07-01 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 § 28.13 Analysis and indexing of DNA samples. (a) The Federal Bureau...

  13. Atrazine, triketone herbicides, and their degradation products in sediment, soil and surface water samples in Poland.

    PubMed

    Barchanska, Hanna; Sajdak, Marcin; Szczypka, Kornelia; Swientek, Angelika; Tworek, Martyna; Kurek, Magdalena

    2017-01-01

    The aim of this study was to monitor the sediment, soil and surface water contamination with selected popular triketone herbicides (mesotrione (MES) and sulcotrione(SUL)), atrazine (ATR) classified as a possible carcinogen and endocrine disrupting chemical, as well as their degradation products, in Silesia (Poland). Seventeen sediment samples, 24 soil samples, and 64 surface water samples collected in 2014 were studied. After solid-liquid extraction (SLE) and solid phase extraction (SPE), analytes were determined by high-performance liquid chromatography (HPLC) with diode array detection (DAD). Ten years after the withdrawal from the use, ATR was not detected in any of the collected samples; however, its degradation products are still present in 41 % of sediment, 71 % of soil, and 8 % of surface water samples. SUL was determined in 85 % of soil samples; its degradation product (2-chloro-4-(methylosulfonyl) benzoic acid (CMBA)) was present in 43 % of soil samples. In 17 % of sediment samples, CMBA was detected. Triketones were detected occasionally in surface water samples. The chemometric analysis (clustering analysis (CA), single-factor analysis of variance (ANOVA), N-Way ANOVA) was applied to find relations between selected soil and sediment parameters and herbicides concentration. In neither of the studied cases a statistically significant relationship between the concentrations of examined herbicides, their degradation products and soil parameters (organic carbon (OC), pH) was observed.

  14. Validated methodology for quantifying infestation levels of dreissenid mussels in environmental DNA (eDNA) samples

    PubMed Central

    Peñarrubia, Luis; Alcaraz, Carles; Vaate, Abraham bij de; Sanz, Nuria; Pla, Carles; Vidal, Oriol; Viñas, Jordi

    2016-01-01

    The zebra mussel (Dreissena polymorpha Pallas, 1771) and the quagga mussel (D. rostriformis Deshayes, 1838) are successful invasive bivalves with substantial ecological and economic impacts in freshwater systems once they become established. Since their eradication is extremely difficult, their detection at an early stage is crucial to prevent spread. In this study, we optimized and validated a qPCR detection method based on the histone H2B gene to quantify combined infestation levels of zebra and quagga mussels in environmental DNA samples. Our results show specific dreissenid DNA present in filtered water samples for which microscopic diagnostic identification for larvae failed. Monitoring a large number of locations for invasive dreissenid species based on a highly specific environmental DNA qPCR assay may prove to be an essential tool for management and control plans focused on prevention of establishment of dreissenid mussels in new locations. PMID:27966602

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

    PubMed

    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

    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 α subunit α4 s/PSMA8 and/or the catalytic β 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.

  16. Forensic DNA typing strategy of degraded DNA on discarded cigarette ends using the AmpFℓSTR® Identifiler®, Identifiler® Plus and MiniFiler™ PCR amplification kits.

    PubMed

    Ip, Stephen C Y; Lin, Sze-wah; Li, Christina; Lai, Kam-ming

    2014-07-01

    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™ AmpFℓSTR® 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™.

  17. Degradation of DNA into 5'-monodeoxyribonucleotides in the presence of Mn(2+) ions.

    PubMed

    Maeda, Hidekatsu; Wada, Shinya; Ikeguchi, Masamichi; Minoura, Norihiko; Ueki, Shouji; Arata, Toshiaki

    2007-11-01

    DNA is known to be aggregated by metal ions including Mn(2+) ions, but analysis of the aggregation process from a chemical viewpoint, which means identification of the product yielded during the process, has not been performed yet. On examination of the kinds of degraded materials that were in the supernatant obtained on centrifugation of a DNA mixture aggregated under conditions of 10 mM Mn(2+) ions ([Mn]/[P] = 46.3) at 70 degrees C for 1 h, the degradation products were found to be dAMP, dCMP, dGMP, and TMP. These dNMPs were purified by HPLC on TSKgel ODS-80Ts and identified by LC-TOF/MS. The degradation activity was lost on pretreatment of the DNA with a phenol-chloroform mixture, and the activity was recovered by pretreatment with a mixture of DMSO and a buffer containing surfactants. Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), and Cd(2+), as transition element metal ions, were effective as to the degradation into dNMP. Mg(2+), Ca(2+), Sr(2+), and Ba(2+), as alkali earth element metal ions, were not effective as to the degradation. Monovalent anions such as Cl(-), CH(3)OO(-), and NO(3)(-) were found to increase the degradation rate. Sixty mug of the 120 mug of the starting DNA in 450 mul was degraded into dNMP on reaction for 1 h in the presence of 100 mM NaCl and 10 mM Mn(2+) ions. In this process, aggregation did not occur, and thus was not considered to be necessary for degradation. The degradation was found not to occur at pH 7.0, and to be very sensitive to pH. The OH(-) ion should have a critical role in cleavage of the phosphodiester linkages in this case. The dNMP obtained in the degradation process was found to be only 5'-NMP, based on the H(1)NMR spectra. This prosess should prove to be a new process for the production of 5'-dNMP in addtion to the exonuclease.

  18. Dual-degradable disulfide-containing PEI-Pluronic/DNA polyplexes: transfection efficiency and balancing protection and DNA release.

    PubMed

    Zhang, Lifen; Chen, Zhenzhen; Li, Yanfeng

    2013-01-01

    Polymeric gene-delivery vectors to achieve lack of toxicity and a balance between protection and DNA release remains a formidable challenge. Incorporating intracellular environment-responsive degradable bonds is an appreciable step toward developing safer transfection agents. In this study, novel, dual-degradable polycation copolymers (Pluronic-diacrylate [PA]-polyethyleneimine [PEI]-SS) were synthesized through the addition of low molecular weight (800 Da) PEI cross-linked with SS (PEI-SS) to PA. Three PA-PEI-SS copolymers (PA-PEI-SS1, 2, and 3) with different PEI-SS to Pluronic molar ratios were investigated and found to strongly condense plasmid DNA into positively charged nanoparticles with an average particle size of approximately 200 nm and to possess higher stability against DNase I digestion and sodium heparin. Disulfide and ester bonds of the copolymers were susceptible to intracellular redox conditions. In vitro experiments demonstrated that the PA-PEI-SS copolymers had significantly lower cytotoxicity and higher transfection efficiency in both BGC-823 and 293T cell lines than the controls of degradable PEI-SS and nondegradable 25 kDa PEI. Transfection activity was influenced by the PEI-SS content in the polymers and PA-PEI-SS1 showed the highest efficiency of the three copolymers. These studies suggest that these dual-degradable copolymers could be used as potential biocompatible gene delivery carriers.

  19. The Effect of Geographical Scale of Sampling on DNA Barcoding

    PubMed Central

    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.

    2012-01-01

    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

  20. Development of an Alu-based, QSY 7-labeled primer PCR method for quantitation of human DNA in forensic samples.

    PubMed

    Nicklas, Janice A; Buel, Eric

    2003-03-01

    Determining the amount of human DNA extracted from a crime scene sample is an important step in DNA profiling. The forensic community relies almost entirely upon a technique (slot blot) to quantitate human DNA that is imprecise, time consuming, and labor intensive. This paper describes the development of a new technique based on PCR amplification of a repetitive Alu sequence. Specific primers were used to amplify a 124-bp fragment of Alu sequence; amplification was detected by SYBR Green I staining in a fluorescent plate reader. To reduce background in the plate reader assay, QSY-7 labeled primers were utilized. The assay was tested on animal DNAs, human blood spots, mock crime samples, and degraded DNA in comparison with the slot blot technique. The QSY Alu assay has a dynamic range of 10 ng to 10 pg, and is sensitive, specific, fast, quantitative, and comparable in cost to the slot blot assay.

  1. Comparison of DNA preservation methods for environmental bacterial community samples

    USGS Publications Warehouse

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

    2013-01-01

    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.

  2. Detection of Invasive Mosquito Vectors Using Environmental DNA (eDNA) from Water Samples

    PubMed Central

    Schneider, Judith; Valentini, Alice; Dejean, Tony; Montarsi, Fabrizio; Taberlet, Pierre

    2016-01-01

    Repeated introductions and spread of invasive mosquito species (IMS) have been recorded on a large scale these last decades worldwide. In this context, members of the mosquito genus Aedes can present serious risks to public health as they have or may develop vector competence for various viral diseases. While the Tiger mosquito (Aedes albopictus) is a well-known vector for e.g. dengue and chikungunya viruses, the Asian bush mosquito (Ae. j. japonicus) and Ae. koreicus have shown vector competence in the field and the laboratory for a number of viruses including dengue, West Nile fever and Japanese encephalitis. Early detection and identification is therefore crucial for successful eradication or control strategies. Traditional specific identification and monitoring of different and/or cryptic life stages of the invasive Aedes species based on morphological grounds may lead to misidentifications, and are problematic when extensive surveillance is needed. In this study, we developed, tested and applied an environmental DNA (eDNA) approach for the detection of three IMS, based on water samples collected in the field in several European countries. We compared real-time quantitative PCR (qPCR) assays specific for these three species and an eDNA metabarcoding approach with traditional sampling, and discussed the advantages and limitations of these methods. Detection probabilities for eDNA-based approaches were in most of the specific comparisons higher than for traditional survey and the results were congruent between both molecular methods, confirming the reliability and efficiency of alternative eDNA-based techniques for the early and unambiguous detection and surveillance of invasive mosquito vectors. The ease of water sampling procedures in the eDNA approach tested here allows the development of large-scale monitoring and surveillance programs of IMS, especially using citizen science projects. PMID:27626642

  3. Detection of Invasive Mosquito Vectors Using Environmental DNA (eDNA) from Water Samples.

    PubMed

    Schneider, Judith; Valentini, Alice; Dejean, Tony; Montarsi, Fabrizio; Taberlet, Pierre; Glaizot, Olivier; Fumagalli, Luca

    2016-01-01

    Repeated introductions and spread of invasive mosquito species (IMS) have been recorded on a large scale these last decades worldwide. In this context, members of the mosquito genus Aedes can present serious risks to public health as they have or may develop vector competence for various viral diseases. While the Tiger mosquito (Aedes albopictus) is a well-known vector for e.g. dengue and chikungunya viruses, the Asian bush mosquito (Ae. j. japonicus) and Ae. koreicus have shown vector competence in the field and the laboratory for a number of viruses including dengue, West Nile fever and Japanese encephalitis. Early detection and identification is therefore crucial for successful eradication or control strategies. Traditional specific identification and monitoring of different and/or cryptic life stages of the invasive Aedes species based on morphological grounds may lead to misidentifications, and are problematic when extensive surveillance is needed. In this study, we developed, tested and applied an environmental DNA (eDNA) approach for the detection of three IMS, based on water samples collected in the field in several European countries. We compared real-time quantitative PCR (qPCR) assays specific for these three species and an eDNA metabarcoding approach with traditional sampling, and discussed the advantages and limitations of these methods. Detection probabilities for eDNA-based approaches were in most of the specific comparisons higher than for traditional survey and the results were congruent between both molecular methods, confirming the reliability and efficiency of alternative eDNA-based techniques for the early and unambiguous detection and surveillance of invasive mosquito vectors. The ease of water sampling procedures in the eDNA approach tested here allows the development of large-scale monitoring and surveillance programs of IMS, especially using citizen science projects.

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

    PubMed

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

    2005-12-28

    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.

  5. FBW7 regulates DNA interstrand cross-link repair by modulating FAAP20 degradation

    PubMed Central

    Wang, Jingming; Jo, Ukhyun; Joo, So Young; Kim, Hyungjin

    2016-01-01

    Mutations that deregulate protein degradation lead to human malignancies. The SCF ubiquitin E3 ligase complex degrades key oncogenic regulators, thereby limiting their oncogenic potential. FBW7 is a substrate recognition subunit of SCFFBW7 and is among the most commonly mutated ubiquitin-proteasome system proteins in cancer. FBW7-mutated cancer cells display increased genome instability, but the molecular mechanism by which FBW7 preserves genome integrity remains elusive. Here, we demonstrate that SCFFBW7 regulates the stability of FAAP20, a critical component of the Fanconi anemia (FA) DNA interstrand cross-link (ICL) repair pathway. Phosphorylation of the FAAP20 degron motif by GSK3β provides a platform for recognition and polyubiquitination of FAAP20 by FBW7, and its subsequent degradation by the proteasome. Accordingly, enhanced GSK3β-FBW7 signaling disrupts the FA pathway. In cells expressing non-phosphorylatable FAAP20 mutant, the turnover of its binding partner, FANCA, is deregulated in the chromatin during DNA ICL repair, and the FA pathway is compromised. We propose that FAAP20 degradation, which is prompted by its phosphorylation, controls the dynamics of the FA core complex required for completing DNA ICL repair. Together, this study provides insights into how FBW7-mediated proteolysis regulates genome stability and how its deregulation is associated with tumorigenesis. PMID:27232758

  6. Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif.

    PubMed

    Land, Allison M; Wang, Jiayi; Law, Emily K; Aberle, Ryan; Kirmaier, Andrea; Krupp, Annabel; Johnson, Welkin E; Harris, Reuben S

    2015-11-24

    APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy.

  7. Analyzing insulin samples by size-exclusion chromatography: a column degradation study.

    PubMed

    Teska, Brandon M; Kumar, Amit; Carpenter, John F; Wempe, Michael F

    2015-04-01

    Investigating insulin analogs and probing their intrinsic stability at physiological temperature, we observed significant degradation in the size-exclusion chromatography (SEC) signal over a moderate number of insulin sample injections, which generated concerns about the quality of the separations. Therefore, our research goal was to identify the cause(s) for the observed signal degradation and attempt to mitigate the degradation in order to extend SEC column lifespan. In these studies, we used multiangle light scattering, nuclear magnetic resonance, and gas chromatography-mass spectrometry methods to evaluate column degradation. The results from these studies illustrate: (1) that zinc ions introduced by the insulin product produced the observed column performance issues; and (2) that including ethylenediaminetetraacetic acid, a zinc chelator, in the mobile phase helped to maintain column performance.

  8. A rapid wire-based sampling method for DNA profiling.

    PubMed

    Chen, Tong; Catcheside, David E A; Stephenson, Alice; Hefford, Chris; Kirkbride, K Paul; Burgoyne, Leigh A

    2012-03-01

    This paper reports the results of a commission to develop a field deployable rapid short tandem repeat (STR)-based DNA profiling system to enable discrimination between tissues derived from a small number of individuals. Speed was achieved by truncation of sample preparation and field deployability by use of an Agilent 2100 Bioanalyser(TM). Human blood and tissues were stabbed with heated stainless steel wire and the resulting sample dehydrated with isopropanol prior to direct addition to a PCR. Choice of a polymerase tolerant of tissue residues and cycles of amplification appropriate for the amount of template expected yielded useful profiles with a custom-designed quintuplex primer set suitable for use with the Bioanalyser(TM). Samples stored on wires remained amplifiable for months, allowing their transportation unrefrigerated from remote locations to a laboratory for analysis using AmpFlSTR(®) Profiler Plus(®) without further processing. The field system meets the requirements for discrimination of samples from small sets and retains access to full STR profiling when required.

  9. Solving practical problems in environmental sampling for chemical agents and their degradation compounds

    SciTech Connect

    Williams, K.E.; Sheely, M.V.

    1995-06-01

    The analyses of environmental samples for chemical agent degradation products were conducted using analytical test methods designed for evaluation of solid waste samples. All methods are found in the 3rd Edition of EPA`s compendium of analytical methods (SW-846) dated July 1992. These EPA methods are recommended for compliance testing required by the Resource Conservation and Recovery Act (RCRA) and are routinely used for the analysis of environmental samples. In the past several years, these same methods were used to support the survey of areas suspected of having chemical agent or chemical agent degradation compound contamination. An overview is presented of the U.S. Army Center for Health Promotion and Preventive Medicine`s (previously the U.S. Army Environmental Hygiene Agency) involvement with the analysis of samples for chemical agents and their degradation compounds collected from sites such as Tooele Army Depot, Rocky Mt. Arsenal, Newport Army Depot, Johnston Island, and Spring Valley, (a residential site near American University in Washington D.C.) Discussed are practical problems encountered during a quick response of a non-surety laboratory to analyze environmental samples for agents and their degradation compounds.

  10. Identification of a new degradation product of the antifouling agent Irgarol 1051 in natural samples

    USGS Publications Warehouse

    Ferrer, I.; Barcelo, D.

    2001-01-01

    A main degradation product of Irgarol [2-(methylthio)-4-(tert-butylamino)-6-(cyclopropylamino)-s-triazine], one of the most widely used compounds in antifouling paints, was detected at trace levels in seawater and sediment samples collected from several marinas on the Mediterranean coast. This degradation product was identified as 2-methylthio-4-tert-butylamino-s-triazine. The unequivocal identification of this compound in seawater samples was carried out by solid-phase extraction (SPE) coupled on-line with liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS). SPE was carried out by passing 150 ml of seawater sample through a cartridge containing a polymeric phase (PLRP-s), with recoveries ranging from 92 to 108% (n=5). Using LC-MS detection in positive ion mode, useful structural information was obtained by increasing the fragmentor voltage, thus permitting the unequivocal identification of this compound in natural samples. Method detection limits were in the range of 0.002 to 0.005 ??g/l. Overall, the combination of on-line SPE and LC-APCI-MS represents an important advance in environmental analysis of herbicide degradation products in seawater, since it demonstrates that trace amounts of new polar metabolites may be determined rapidly. This paper reports the LC-MS identification of the main degradation product of Irgarol in seawater and sediment samples. ?? 2001 Elsevier Science B.V. All rights reserved.

  11. Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA.

    PubMed

    Asari, Masaru; Watanabe, Satoshi; Matsubara, Kazuo; Shiono, Hiroshi; Shimizu, Keiko

    2009-03-01

    Single nucleotide polymorphism (SNP) is informative for human identification, and much shorter regions are targeted in analysis of biallelic SNP compared with highly polymorphic short tandem repeat (STR). Therefore, SNP genotyping is expected to be more sensitive than STR genotyping of degraded human DNA. To achieve simple, economical, and sensitive SNP genotyping for identification of degraded human DNA, we developed 18 loci for a SNP genotyping technique based on the mini-primer allele-specific amplification (ASA) combined with universal reporter primers (URP). The URP/ASA-based genotyping consisted of two amplifications followed by detection using capillary electrophoresis. The sizes of the target genome fragments ranged from 40 to 67bp in length. In the Japanese population, the frequencies of minor alleles of 18 SNPs ranged from 0.36 to 0.50, and these SNPs are informative for identification. The success rate of SNP genotyping was much higher than that of STR genotyping of artificially degraded DNA. Moreover, we applied this genotyping method to case samples and showed successful SNP genotyping of severely degraded DNA from a 4-year buffered formalin-fixed tissue sample for human identification.

  12. Relative rates of repair of single-strand breaks and postirradiation DNA degradation in normal and induced cells of Escherichia coli.

    PubMed Central

    Pollard, E C; Fugate, J K

    1978-01-01

    Labeled DNA from irradiated Excherichia coli cells has been studied on an alkaline sucrose gradient without acid precipitation of the DNA. This enables the observation of both DNA repair and DNA degradation. The use of a predose of ultraviolet light (UV) causes induction of an inhibitor of postirradiation DNA degradation in lex+ strains. The effect of this induction on both the repair of single-strand breaks and DNA degradation has been followed in strains WU3610 (uvr+) and WU3610-89 (uvr-). The repair process is more rapid than the degradation, and when degradation is inhibited more repair is apparent. Cells that are lex- (Bs-1 and AB2474) cannot be induced for inhibition of degradation. Nevertheless, by observation at short times repair can be seen clearly. This repaired DNA is degraded, suggesting that the signal for DNA degradation is not a single-strand break. PMID:365253

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

    PubMed

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

    2013-11-01

    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.

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

    PubMed Central

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

    2013-01-01

    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 13C-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

  15. HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages

    PubMed Central

    Lahouassa, Hichem; Blondot, Marie-Lise; Chauveau, Lise; Chougui, Ghina; Morel, Marina; Leduc, Marjorie; Guillonneau, François; Ramirez, Bertha Cecilia; Schwartz, Olivier; Margottin-Goguet, Florence

    2016-01-01

    Viruses often interfere with the DNA damage response to better replicate in their hosts. The human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) protein has been reported to modulate the activity of the DNA repair structure-specific endonuclease subunit (SLX4) complex and to promote cell cycle arrest. Vpr also interferes with the base-excision repair pathway by antagonizing the uracil DNA glycosylase (Ung2) enzyme. Using an unbiased quantitative proteomic screen, we report that Vpr down-regulates helicase-like transcription factor (HLTF), a DNA translocase involved in the repair of damaged replication forks. Vpr subverts the DDB1–cullin4-associated-factor 1 (DCAF1) adaptor of the Cul4A ubiquitin ligase to trigger proteasomal degradation of HLTF. This event takes place rapidly after Vpr delivery to cells, before and independently of Vpr-mediated G2 arrest. HLTF is degraded in lymphocytic cells and macrophages infected with Vpr-expressing HIV-1. Our results reveal a previously unidentified strategy for HIV-1 to antagonize DNA repair in host cells. PMID:27114546

  16. Microbial diversity in fecal samples depends on DNA extraction method: easyMag DNA extraction compared to QIAamp DNA stool mini kit extraction

    PubMed Central

    2014-01-01

    Background There are challenges, when extracting bacterial DNA from specimens for molecular diagnostics, since fecal samples also contain DNA from human cells and many different substances derived from food, cell residues and medication that can inhibit downstream PCR. The purpose of the study was to evaluate two different DNA extraction methods in order to choose the most efficient method for studying intestinal bacterial diversity using Denaturing Gradient Gel Electrophoresis (DGGE). Findings In this study, a semi-automatic DNA extraction system (easyMag®, BioMérieux, Marcy I’Etoile, France) and a manual one (QIAamp DNA Stool Mini Kit, Qiagen, Hilden, Germany) were tested on stool samples collected from 3 patients with Inflammatory Bowel disease (IBD) and 5 healthy individuals. DNA extracts obtained by the QIAamp DNA Stool Mini Kit yield a higher amount of DNA compared to DNA extracts obtained by easyMag® from the same fecal samples. Furthermore, DNA extracts obtained using easyMag® seemed to contain inhibitory compounds, since in order to perform a successful PCR-analysis, the sample should be diluted at least 10 times. DGGE performed on PCR from DNA extracted by QIAamp DNA Stool Mini Kit DNA was very successful. Conclusion QIAamp DNA Stool Mini Kit DNA extracts are optimal for DGGE runs and this extraction method yields a higher amount of DNA compared to easyMag®. PMID:24447346

  17. Microbial degradation of gasoline in soil: Effect of season of sampling.

    PubMed

    Turner, D A; Pichtel, J; Rodenas, Y; McKillip, J; Goodpaster, J V

    2015-06-01

    In cases where fire debris contains soil, microorganisms can rapidly and irreversibly alter the chemical composition of any ignitable liquid residue that may be present. In this study, differences in microbial degradation due to the season in which the sample is collected was examined. Soil samples were collected from the same site during Fall, Winter, Spring and Summer and the degradation of gasoline was monitored over 30 days. Predominant viable bacterial populations enumerated using real-time PCR and reverse transcriptase polymerase chain reaction (RT-PCR) enumeration revealed the predominant viable bacterial genera to be Alcaligenes, Bacillus, and Flavobacterium. Overall, the compounds most vulnerable to microbial degradation are the n-alkanes, followed by the mono-substituted alkylbenzenes (e.g., toluene, ethylbenzene, propylbenzene and isopropylbenzene). Benzaldehyde (a degradation product of toluene) was also identified as a marker for the extent of biodegradation. Ultimately, it was determined that soil collected during an unusually hot and dry summer exhibited the least degradation with little to no change in gasoline for up to 4 days, readily detectable n-alkanes for up to 7 days and relatively high levels of resilient compounds such as o-xylene, p-xylene and 1,3,5-trimethylbenzene. These results demonstrate, however, that prompt preservation and/or analysis of soil evidence is required in order to properly classify an ignitable liquid residue.

  18. Comparison of four commercial DNA extraction kits for the recovery of Bacillus spp. spore DNA from spiked powder samples.

    PubMed

    Mölsä, Markos; Kalin-Mänttäri, Laura; Tonteri, Elina; Hemmilä, Heidi; Nikkari, Simo

    2016-09-01

    Bacillus spp. include human pathogens such as Bacillus anthracis, the causative agent of anthrax and a biothreat agent. Bacillus spp. form spores that are physically highly resistant and may remain active over sample handling. We tested four commercial DNA extraction kits (QIAamp DNA Mini Kit, RTP Pathogen Kit, ZR Fungal/Bacterial DNA MiniPrep, and genesig Easy DNA/RNA Extraction kit) for sample inactivation and DNA recovery from two powders (icing sugar and potato flour) spiked with Bacillus thuringiensis spores. The DNA was analysed using a B. thuringiensis-specific real-time PCR assay. The detection limit was 3×10(1)CFU of spiked B. thuringiensis spores with the QIAamp DNA Mini, RTP Pathogen, and genesig Easy DNA/RNA Extraction kits, and 3×10(3)CFU with the ZR Fungal/Bacterial DNA MiniPrep kit. The results showed that manual extraction kits are effective and safe for fast and easy DNA extraction from powder samples even in field conditions. Adding a DNA filtration step to the extraction protocol ensures the removal of Bacillus spp. spores from DNA samples without affecting sensitivity.

  19. DDB2 association with PCNA is required for its degradation after UV-induced DNA damage.

    PubMed

    Cazzalini, Ornella; Perucca, Paola; Mocchi, Roberto; Sommatis, Sabrina; Prosperi, Ennio; Stivala, Lucia Anna

    2014-01-01

    DDB2 is a protein playing an essential role in the lesion recognition step of the global genome sub-pathway of nucleotide excision repair (GG-NER) process. Among the proteins involved in the DNA damage response, p21(CDKN1A) (p21) has been reported to participate in NER, but also to be removed by proteolytic degradation, thanks to its association with PCNA. DDB2 is involved in the CUL4-DDB1 complex mediating p21 degradation; however, the direct interaction between DDB2, p21 and PCNA has been never investigated. Here, we show that DDB2 co-localizes with PCNA and p21 at local UV-induced DNA-damage sites, and these proteins co-immunoprecipitate in the same complex. In addition, we provide evidence that p21 is not able to bind directly DDB2, but, to this end, the presence of PCNA is required. Direct physical association of recombinant DDB2 protein with PCNA is mediated by a conserved PIP-box present in the N-terminal region of DDB2. Mutation of the PIP-box resulted in the loss of protein interaction. Interestingly, the same mutation, or depletion of PCNA by RNA interference, greatly impaired DDB2 degradation induced by UV irradiation. These results indicate that DDB2 is a PCNA-binding protein, and that this association is required for DDB2 proteolytic degradation.

  20. Major degradable polycations as carriers for DNA and siRNA.

    PubMed

    Islam, Mohammad Ariful; Park, Tae-Eun; Singh, Bijay; Maharjan, Sushila; Firdous, Jannatul; Cho, Myung-Haing; Kang, Sang-Kee; Yun, Cheol-Heui; Choi, Yun-Jaie; Cho, Chong-Su

    2014-11-10

    Non-viral gene delivery systems are one of the most potential alternatives to viral vectors because of their less immunogenicity, less toxicity and easy productivity in spite of their low capacity of gene transfection using DNA or silencing using siRNA compared to that of viral vectors. Among non-viral systems, the polycationic derivatives are the most popular gene carriers since they can effectively condense nucleic acids to transfer into the cells, especially the polyethylenimine (PEI) which has been used as a golden standard polymer owing to its high buffering ability for endosomal escape of gene to be expressed. However, PEI has severe problems for its toxicity due to the high positive charge density and non-degradability although the toxicity of PEI depends on its molecular weight (MW) and structure. Therefore, a considerable attention has been paid on synthesis of degradable PEI derivatives using low MW one because low MW PEI is much less toxic than high MW PEI. Other degradable polycationic gene carriers such as polyamidoamines (PAA) and cyclodextrin (CD)-based polycations are also in a significant interest because of their high transfection efficiency with low toxicity. This review in detail explains the recent developments on these three major degradable polycations as promising carriers for deoxyribonucleic acid (DNA) and small interfering RNA (siRNA).

  1. A method for DNA and RNA co-extraction for use on forensic samples using the Promega DNA IQ™ system.

    PubMed

    Bowden, Anna; Fleming, Rachel; Harbison, SallyAnn

    2011-01-01

    The use of messenger RNA profiling to identify the origin of biological samples (e.g. blood, semen and saliva) from crime scenes is now at the stage of being implemented into routine forensic casework. We report on the successful modification of the Promega DNA IQ™ system to enable co-extraction of DNA and RNA from the same sample without compromising the potential DNA profile. Using the protocol in our laboratory for extracting DNA using the DNA IQ™ system combined with the Zymo Research Mini RNA Isolation Kit™ II we demonstrate the simultaneous co-extraction of DNA and RNA from the same sample for routine DNA and mRNA profiling for the identification of both the individual and the biological stain.

  2. Performance verification of the Maxwell 16 Instrument and DNA IQ Reference Sample Kit for automated DNA extraction of known reference samples.

    PubMed

    Krnajski, Z; Geering, S; Steadman, S

    2007-12-01

    Advances in automation have been made for a number of processes conducted in the forensic DNA laboratory. However, because most robotic systems are designed for high-throughput laboratories batching large numbers of samples, smaller laboratories are left with a limited number of cost-effective options for employing automation. The Maxwell 16 Instrument and DNA IQ Reference Sample Kit marketed by Promega are designed for rapid, automated purification of DNA extracts from sample sets consisting of sixteen or fewer samples. Because the system is based on DNA capture by paramagnetic particles with maximum binding capacity, it is designed to generate extracts with yield consistency. The studies herein enabled evaluation of STR profile concordance, consistency of yield, and cross-contamination performance for the Maxwell 16 Instrument. Results indicate that the system performs suitably for streamlining the process of extracting known reference samples generally used for forensic DNA analysis and has many advantages in a small or moderate-sized laboratory environment.

  3. Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena

    PubMed Central

    Schoeberl, Ursula E.; Kurth, Henriette M.; Noto, Tomoko; Mochizuki, Kazufumi

    2012-01-01

    The ciliated protozoan Tetrahymena undergoes extensive programmed DNA elimination when the germline micronucleus produces the new macronucleus during sexual reproduction. DNA elimination is epigenetically controlled by DNA sequences of the parental macronuclear genome, and this epigenetic regulation is mediated by small RNAs (scan RNAs [scnRNAs]) of ∼28–30 nucleotides that are produced and function by an RNAi-related mechanism. Here, we examine scnRNA production and turnover by deep sequencing. scnRNAs are produced exclusively from the micronucleus and nonhomogeneously from a variety of chromosomal locations. scnRNAs are preferentially derived from the eliminated sequences, and this preference is mainly determined at the level of transcription. Despite this bias, a significant fraction of scnRNAs is also derived from the macronuclear-destined sequences, and these scnRNAs are degraded during the course of sexual reproduction. These results indicate that the pattern of DNA elimination in the new macronucleus is shaped by the biased transcription in the micronucleus and the selective degradation of scnRNAs in the parental macronucleus. PMID:22855833

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

    PubMed

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

    2014-01-05

    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.

  5. Fast identification of microplastics in complex environmental samples by a thermal degradation method.

    PubMed

    Dümichen, Erik; Eisentraut, Paul; Bannick, Claus Gerhard; Barthel, Anne-Kathrin; Senz, Rainer; Braun, Ulrike

    2017-05-01

    In order to determine the relevance of microplastic particles in various environmental media, comprehensive investigations are needed. However, no analytical method exists for fast identification and quantification. At present, optical spectroscopy methods like IR and RAMAN imaging are used. Due to their time consuming procedures and uncertain extrapolation, reliable monitoring is difficult. For analyzing polymers Py-GC-MS is a standard method. However, due to a limited sample amount of about 0.5 mg it is not suited for analysis of complex sample mixtures like environmental samples. Therefore, we developed a new thermoanalytical method as a first step for identifying microplastics in environmental samples. A sample amount of about 20 mg, which assures the homogeneity of the sample, is subjected to complete thermal decomposition. The specific degradation products of the respective polymer are adsorbed on a solid-phase adsorber and subsequently analyzed by thermal desorption gas chromatography mass spectrometry. For certain identification, the specific degradation products for the respective polymer were selected first. Afterwards real environmental samples from the aquatic (three different rivers) and the terrestrial (bio gas plant) systems were screened for microplastics. Mainly polypropylene (PP), polyethylene (PE) and polystyrene (PS) were identified for the samples from the bio gas plant and PE and PS from the rivers. However, this was only the first step and quantification measurements will follow.

  6. Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments

    SciTech Connect

    Walia, S.; Khan, A.; Rosenthal, N. )

    1990-01-01

    Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community.

  7. In-solution hybridization for mammalian mitogenome enrichment: pros, cons and challenges associated with multiplexing degraded DNA.

    PubMed

    Hawkins, Melissa T R; Hofman, Courtney A; Callicrate, Taylor; McDonough, Molly M; Tsuchiya, Mirian T N; Gutiérrez, Eliécer E; Helgen, Kristofer M; Maldonado, Jesus E

    2016-09-01

    Here, we present a set of RNA-based probes for whole mitochondrial genome in-solution enrichment, targeting a diversity of mammalian mitogenomes. This probes set was designed from seven mammalian orders and tested to determine the utility for enriching degraded DNA. We generated 63 mitogenomes representing five orders and 22 genera of mammals that yielded varying coverage ranging from 0 to >5400X. Based on a threshold of 70% mitogenome recovery and at least 10× average coverage, 32 individuals or 51% of samples were considered successful. The estimated sequence divergence of samples from the probe sequences used to construct the array ranged up to nearly 20%. Sample type was more predictive of mitogenome recovery than sample age. The proportion of reads from each individual in multiplexed enrichments was highly skewed, with each pool having one sample that yielded a majority of the reads. Recovery across each mitochondrial gene varied with most samples exhibiting regions with gaps or ambiguous sites. We estimated the ability of the probes to capture mitogenomes from a diversity of mammalian taxa not included here by performing a clustering analysis of published sequences for 100 taxa representing most mammalian orders. Our study demonstrates that a general array can be cost and time effective when there is a need to screen a modest number of individuals from a variety of taxa. We also address the practical concerns for using such a tool, with regard to pooling samples, generating high quality mitogenomes and detail a pipeline to remove chimeric molecules.

  8. Performance degradation of a Michelson interferometer due to random sampling errors.

    PubMed

    Cohen, D L

    1999-01-01

    The performance of a standard Michelson interferometer is degraded by disturbances that cause the interferogram signal to be sampled at nonconstant time intervals. A formula that shows how the power spectrum of the random disturbances interacts with the signal to contaminate different regions of the measured spectrum is derived for the spectral noise. The sampling noise does not look conventionally noiselike because it is correlated over large regions of the measured spectrum, and adjustment of the unbalanced background interferogram to match the size of the balanced background interferogram minimizes the sampling-noise amplitude.

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

    PubMed

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

    2014-03-06

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

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

    PubMed Central

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

    2014-01-01

    Ancient human remains of paleopathological interest typically contain highly degraded DNA in which pathogenic taxa are often minority components, making sequence-based metagenomic characterization costly. Microarrays may hold a potential solution to these challenges, offering a rapid, affordable, and highly informative snapshot of microbial diversity in complex samples without the lengthy analysis and/or high cost associated with high-throughput sequencing. Their versatility is well established for modern clinical specimens, but they have yet to be applied to ancient remains. Here we report bacterial profiles of archaeological and historical human remains using the Lawrence Livermore Microbial Detection Array (LLMDA). The array successfully identified previously-verified bacterial human pathogens, including Vibrio cholerae (cholera) in a 19th century intestinal specimen and Yersinia pestis (“Black Death” plague) in a medieval tooth, which represented only minute fractions (0.03% and 0.08% alignable high-throughput shotgun sequencing reads) of their respective DNA content. This demonstrates that the LLMDA can identify primary and/or co-infecting bacterial pathogens in ancient samples, thereby serving as a rapid and inexpensive paleopathological screening tool to study health across both space and time. PMID:24603850

  11. Metal activation of synthetic and degradative activities of phi 29 DNA polymerase, a model enzyme for protein-primed DNA replication.

    PubMed

    Esteban, J A; Bernad, A; Salas, M; Blanco, L

    1992-01-21

    Analysis of metal activation on the synthetic and degradative activities of phi 29 DNA polymerase was carried out in comparison with T4 DNA polymerase and Escherichia coli DNA polymerase I (Klenow fragment). In the three DNA polymerases studied, both the polymerization and the 3'----5' exonuclease activity had clear differences in their metal ion requirements. The results obtained support the existence of independent metal binding sites for the synthetic and degradative activities of phi 29 DNA polymerase, according with the distant location of catalytic domains (N-terminal for the 3'----5' exonuclease and C-terminal for DNA polymerization) proposed for both Klenow fragment and phi 29 DNA polymerase. Furthermore, DNA competition experiments using phi 29 DNA polymerase suggested that the main differences observed in the metal usage to activate polymerization may be the consequence of metal-induced changes in the enzyme-DNA interactions, whose strength distinguishes processive and nonprocessive DNA polymerases. Interestingly, the initiation of DNA polymerization using a protein as a primer, a special synthetic activity carried out by phi 29 DNA polymerase, exhibited a strong preference for Mn2+ as metal activator. The molecular basis for this preference is mainly the result of a large increase in the affinity for dATP.

  12. Ozone degradation of residual carbon in biological samples using microwave irradiation.

    PubMed

    Jiang, W; Chalk, S J; Kingston, H M

    1997-03-01

    In an attempt to produce complete oxidation of a biological matrix, bovine liver, ozone was investigated as an additional, potentially non-contaminating, oxidizing reagent after nitric acid digestion. Experiments were carried out to determine the decomposition efficiency of residual carbon species, primarily o-, m- and p-nitrobenzoic acids (NBAs), using ozone. The NBAs were degraded by purging sample digests with ozone, while heating the solutions with microwave energy at atmospheric pressure. The effects of the degradation temperature and solution pH on the ozonation of NBAs were determined. Solid phase extraction (C18) was used to extract NBAs from the acid digestate solutions prior to HPLC analysis. Reversed phase HPLC was used to determine NBA concentrations in digest solutions. After 2.5 h of purging ozone at 80 degrees C, 33.65 +/- 3.80% o-NBA degradation, 19.39 +/- 1.74% m-NBA degradation, and 26.47 +/- 3.36% p-NBA degradation were obtained.

  13. 75 FR 32191 - National Health and Nutrition Examination Survey (NHANES) DNA Samples: Guidelines for Proposals...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-07

    ... Survey (NHANES) DNA Samples: Guidelines for Proposals To Use Samples and Cost Schedule AGENCY: Centers... extensive amount of information collected for the purpose of describing the health of the population, DNA specimens were collected during three NHANES surveys. DNA is available in the form of crude lysates of...

  14. Degradation and detection of transgenic Bacillus thuringiensis DNA and proteins in flour of three genetically modified rice events submitted to a set of thermal processes.

    PubMed

    Wang, Xiaofu; Chen, Xiaoyun; Xu, Junfeng; Dai, Chen; Shen, Wenbiao

    2015-10-01

    This study aimed to investigate the degradation of three transgenic Bacillus thuringiensis (Bt) genes (Cry1Ab, Cry1Ac, and Cry1Ab/Ac) and the corresponding encoded Bt proteins in KMD1, KF6, and TT51-1 rice powder, respectively, following autoclaving, cooking, baking, or microwaving. Exogenous Bt genes were more stable than the endogenous sucrose phosphate synthase (SPS) gene, and short DNA fragments were detected more frequently than long DNA fragments in both the Bt and SPS genes. Autoclaving, cooking (boiling in water, 30 min), and baking (200 °C, 30 min) induced the most severe Bt protein degradation effects, and Cry1Ab protein was more stable than Cry1Ac and Cry1Ab/Ac protein, which was further confirmed by baking samples at 180 °C for different periods of time. Microwaving induced mild degradation of the Bt and SPS genes, and Bt proteins, whereas baking (180 °C, 15 min), cooking and autoclaving led to further degradation, and baking (200 °C, 30 min) induced the most severe degradation. The findings of the study indicated that degradation of the Bt genes and proteins somewhat correlated with the treatment intensity. Polymerase chain reaction, enzyme-linked immunosorbent assay, and lateral flow tests were used to detect the corresponding transgenic components. Strategies for detecting transgenic ingredients in highly processed foods are discussed.

  15. Specific amplification of bacterial DNA by optimized so-called universal bacterial primers in samples rich of plant DNA.

    PubMed

    Dorn-In, Samart; Bassitta, Rupert; Schwaiger, Karin; Bauer, Johann; Hölzel, Christina S

    2015-06-01

    Universal primers targeting the bacterial 16S-rRNA-gene allow quantification of the total bacterial load in variable sample types by qPCR. However, many universal primer pairs also amplify DNA of plants or even of archaea and other eukaryotic cells. By using these primers, the total bacterial load might be misevaluated, whenever samples contain high amounts of non-target DNA. Thus, this study aimed to provide primer pairs which are suitable for quantification and identification of bacterial DNA in samples such as feed, spices and sample material from digesters. For 42 primers, mismatches to the sequence of chloroplasts and mitochondria of plants were evaluated. Six primer pairs were further analyzed with regard to the question whether they anneal to DNA of archaea, animal tissue and fungi. Subsequently they were tested with sample matrix such as plants, feed, feces, soil and environmental samples. To this purpose, the target DNA in the samples was quantified by qPCR. The PCR products of plant and feed samples were further processed for the Single Strand Conformation Polymorphism method followed by sequence analysis. The sequencing results revealed that primer pair 335F/769R amplified only bacterial DNA in samples such as plants and animal feed, in which the DNA of plants prevailed.

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

    USGS Publications Warehouse

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

    2002-01-01

    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.

  17. Modified DOP-PCR for improved STR typing of degraded DNA from human skeletal remains and bloodstains.

    PubMed

    Ambers, Angie; Turnbough, Meredith; Benjamin, Robert; Gill-King, Harrell; King, Jonathan; Sajantila, Antti; Budowle, Bruce

    2016-01-01

    Forensic and ancient DNA samples often are damaged and in limited quantity as a result of exposure to harsh environments and the passage of time. Several strategies have been proposed to address the challenges posed by degraded and low copy templates, including a PCR based whole genome amplification method called degenerate oligonucleotide-primed PCR (DOP-PCR). This study assessed the efficacy of four modified versions of the original DOP-PCR primer that retain at least a portion of the 5' defined sequence and alter the number of bases on the 3' end. The use of each of the four modified primers resulted in improved STR profiles from environmentally-damaged bloodstains, contemporary human skeletal remains, American Civil War era bone samples, and skeletal remains of WWII soldiers over those obtained by previously described DOP-PCR methods and routine STR typing. Additionally, the modified DOP-PCR procedure allows for a larger volume of DNA extract to be used, reducing the need to concentrate the sample and thus mitigating the effects of concurrent concentration of inhibitors.

  18. Assessing uncertainty in DNA evidence caused by sampling effects.

    PubMed

    Curran, J M; Buckleton, J S; Triggs, C M; Weir, B S

    2002-01-01

    Sampling error estimation in forensic DNA testimony was discussed. Is an estimate necessary and how should it be made? The authors find that all modern methods have areas of strength and weakness. The assessment of which is the 'best' is subjective and depends on the performance of the method, the type of problem (criminal work or paternity), the database size and availability of computing software and support. The authors preferred the highest posterior density approach for performance, however the other methods all have areas where their performance is adequate. For single-contributor stains normal approximation methods are suitable, also the bootstrap and the highest posterior density method. For multiple-contributor stains or other complex situations the match probability expressions become quite complex and it may not be possible to derive the necessary variance expressions. The highest posterior density or the bootstrap provide a better general method, with non-zero theta. The size-bias correction and the factor of 10 approaches may be considered acceptable by many forensic scientists as long as their limitations are understood.

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

    PubMed

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

    2013-01-01

    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, γ-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.

  20. Analysis Of Condensate Samples In Support Of The Antifoam Degradation Study

    SciTech Connect

    Hay, M.; Martino, C.

    2016-01-12

    The degradation of Antifoam 747 to form flammable decomposition products has resulted in declaration of a Potential Inadequacy in the Safety Analysis (PISA) for the Defense Waste Processing Facility (DWPF). Savannah River National Laboratory (SRNL) testing with simulants showed that hexamethyldisiloxane (HMDSO), trimethylsilanol (TMS), and 1-propanal are formed in the offgas from the decomposition of the antifoam. A total of ten DWPF condensate samples from Batch 735 and 736 were analyzed by SRNL for three degradation products and additional analytes. All of the samples were analyzed to determine the concentrations of HMDSO, TMS, and propanal. The results of the organic analysis found concentrations for propanal and HMDSO near or below the detection limits for the analysis. The TMS concentrations ranged from below detection to 11 mg/L. The samples from Batch 736 were also analyzed for formate and oxalate anions, total organic carbon, and aluminum, iron, manganese, and silicon. Most of the samples contained low levels of formate and therefore low levels of organic carbon. These two values for each sample show reasonable agreement in most cases. Low levels of all the metals (Al, Fe, Mn, and Si) were present in most of the samples.

  1. ANALYSIS OF CONDENSATE SAMPLES IN SUPPORT OF THE ANTIFOAM DEGRADATION STUDY

    SciTech Connect

    Hay, M.; Martino, C.

    2016-02-29

    The degradation of Antifoam 747 to form flammable decomposition products has resulted in declaration of a Potential Inadequacy in the Safety Analysis (PISA) for the Defense Waste Processing Facility (DWPF). Savannah River National Laboratory (SRNL) testing with simulants showed that hexamethyldisiloxane (HMDSO), trimethylsilanol (TMS), and 1-propanal are formed in the offgas from the decomposition of the antifoam. A total of ten DWPF condensate samples from Batch 735 and 736 were analyzed by SRNL for three degradation products and additional analytes. All of the samples were analyzed to determine the concentrations of HMDSO, TMS, and propanal. The results of the organic analysis found concentrations for propanal and HMDSO near or below the detection limits for the analysis. The TMS concentrations ranged from below detection to 11 mg/L. The samples from Batch 736 were also analyzed for formate and oxalate anions, total organic carbon, and aluminum, iron, manganese, and silicon. Most of the samples contained low levels of formate and therefore low levels of organic carbon. These two values for each sample show reasonable agreement in most cases. Low levels of all the metals (Al, Fe, Mn, and Si) were present in most of the samples.

  2. Presence and potential of cell free DNA in different types of forensic samples.

    PubMed

    Vandewoestyne, Mado; Van Hoofstat, David; Franssen, Aimée; Van Nieuwerburgh, Filip; Deforce, Dieter

    2013-02-01

    Extracellular or cell free DNA has been found to exist in many biological media such as blood and saliva. To check whether cell free DNA is present in the supernatant which is normally discarded during several DNA extraction processes, such as Chelex(®) extraction, DNA profiles of cell pellet and concentrated supernatant from 30 artificial case like samples and from 100 real forensic samples were compared. Presence of cell free DNA was shown in all investigated sample types. Moreover, in some samples additional alleles, not detected during analysis of the cell pellet, were detected, offering valuable information which would normally have been discarded together with the supernatant. The results presented here indicate that cell free DNA deserves further consideration since it has the potential to increase the DNA yield in forensic casework samples in general and in contact traces in particular.

  3. Elimination of bioweapons agents from forensic samples during extraction of human DNA.

    PubMed

    Timbers, Jason; Wilkinson, Della; Hause, Christine C; Smith, Myron L; Zaidi, Mohsin A; Laframboise, Denis; Wright, Kathryn E

    2014-11-01

    Collection of DNA for genetic profiling is a powerful means for the identification of individuals responsible for crimes and terrorist acts. Biologic hazards, such as bacteria, endospores, toxins, and viruses, could contaminate sites of terrorist activities and thus could be present in samples collected for profiling. The fate of these hazards during DNA isolation has not been thoroughly examined. Our goals were to determine whether the DNA extraction process used by the Royal Canadian Mounted Police eliminates or neutralizes these agents and if not, to establish methods that render samples safe without compromising the human DNA. Our results show that bacteria, viruses, and toxins were reduced to undetectable levels during DNA extraction, but endospores remained viable. Filtration of samples after DNA isolation eliminated viable spores from the samples but left DNA intact. We also demonstrated that contamination of samples with some bacteria, endospores, and toxins for longer than 1 h compromised the ability to complete genetic profiling.

  4. Large volume sample stacking in capillary zone electrophoresis for the monitoring of the degradation products of metribuzin in environmental samples.

    PubMed

    Quesada-Molina, Carolina; García-Campaña, Ana M; Del Olmo-Iruela, Laura; Del Olmo, Monsalud

    2007-09-14

    A capillary zone electrophoresis (CZE) method with UV-vis detection has been developed for the simultaneous monitoring of the major degradation products of metribuzin, i.e. deaminometribuzin (DA), deaminodiketometribuzin (DADK) and diketometribuzin (DK). The dissociation acid constants have also been estimated by CE and no significant differences have been observed with the values obtained by applying other techniques. Optimum separation has been achieved in less than 9 min in 40 mM sodium tetraborate buffer, pH 9.5 by applying a voltage of 15kV at 25 degrees C and using p-aminobenzoic acid as internal standard. In order to increase sensitivity, large volume sample stacking (LVSS) with polarity switching has been applied as on-line pre-concentration methodology. Detection limits of 10, 10 and 20 ng/mL for DA, DADK and DK, respectively were obtained. The method has been applied to soil samples, after pressurized liquid extraction (PLE). Samples were extracted at high temperature (103 degrees C and 1500 psi) using methanol as extraction solvent and sodium sulphate as drying agent. This PLE procedure was followed by an off-line pre-concentration and sample clean-up procedure by solid-phase extraction (SPE) using a LiChrolut EN sorbent column. These last two procedures were also suitable for the direct treatment of groundwater samples before CE analysis. The combination of both off-line and on-line pre-concentration procedures provided a significant improvement in sensitivity. LVSS provided pre-concentration factors of 4, 36 and 28 for DK, DA and DADK, respectively and with SPE a pre-concentration of 500-fold for the case of water samples and of 2.5-fold in the case of soil samples was obtained. The method is suitable for the monitoring of these residues in environmental samples with high sensitivity, precision and satisfactory recoveries.

  5. The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform-isoamyl alcohol DNA extraction.

    PubMed

    Renshaw, Mark A; Olds, Brett P; Jerde, Christopher L; McVeigh, Margaret M; Lodge, David M

    2015-01-01

    Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2-week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol-chloroform-isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction.

  6. The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol–chloroform–isoamyl alcohol DNA extraction

    PubMed Central

    Renshaw, Mark A; Olds, Brett P; Jerde, Christopher L; McVeigh, Margaret M; Lodge, David M

    2015-01-01

    Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2-week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol–chloroform–isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction. PMID:24834966

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

    NASA Astrophysics Data System (ADS)

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

    2010-09-01

    Silver "nano-necklaces" and nanoplates in DNA/Tris-EDTA (TE) solution are prepared using hydrothermal method. The nano-necklaces are composed of many spherical silver nanoparticles which are joined together by the DNA chain. Further the transition from silver nano-necklaces to triangular and hexagonal nanoplates is realized based on the degradation of DNA. Transmission electron microscopy, selected area electron diffraction, ultraviolet-visible spectroscopy, X-ray diffraction, and agarose gel electrophoresis are used to characterize silver nanoparticles and the change of DNA structure. The results show that TE acts as not only the buffer solution but also the reducing agent. DNA serves as templates to offer the nucleation sites and induce the growth of silver nanostructures. Hydrothermal process provides high temperature and pressure to activate the reducing property of TE and denature or degrade DNA molecules. The formation mechanism of silver nano-necklaces and nanoplates has also been studied.

  8. Protocol Improvements for Low Concentration DNA-Based Bioaerosol Sampling and Analysis

    PubMed Central

    Ng, Chun Kiat; Miller, Dana; Cao, Bin

    2015-01-01

    Introduction As bioaerosol research attracts increasing attention, there is a need for additional efforts that focus on method development to deal with different environmental samples. Bioaerosol environmental samples typically have very low biomass concentrations in the air, which often leaves researchers with limited options in choosing the downstream analysis steps, especially when culture-independent methods are intended. Objectives This study investigates the impacts of three important factors that can influence the performance of culture-independent DNA-based analysis in dealing with bioaerosol environmental samples engaged in this study. The factors are: 1) enhanced high temperature sonication during DNA extraction; 2) effect of sampling duration on DNA recoverability; and 3) an alternative method for concentrating composite samples. In this study, DNA extracted from samples was analysed using the Qubit fluorometer (for direct total DNA measurement) and quantitative polymerase chain reaction (qPCR). Results and Findings The findings suggest that additional lysis from high temperature sonication is crucial: DNA yields from both high and low biomass samples increased up to 600% when the protocol included 30-min sonication at 65°C. Long air sampling duration on a filter media was shown to have a negative impact on DNA recoverability with up to 98% of DNA lost over a 20-h sampling period. Pooling DNA from separate samples during extraction was proven to be feasible with margins of error below 30%. PMID:26619279

  9. Practical method for extraction of PCR-quality DNA from environmental soil samples.

    PubMed

    Fitzpatrick, Kelly A; Kersh, Gilbert J; Massung, Robert F

    2010-07-01

    Methods for the extraction of PCR-quality DNA from environmental soil samples by using pairs of commercially available kits were evaluated. Coxiella burnetii DNA was detected in spiked soil samples at <1,000 genome equivalents per gram of soil and in 12 (16.4%) of 73 environmental soil samples.

  10. Prediction of people's origin from degraded DNA--presentation of SNP assays and calculation of probability.

    PubMed

    Poetsch, Micaela; Blöhm, Rowena; Harder, Melanie; Inoue, Hiromasa; von Wurmb-Schwark, Nicole; Freitag-Wolf, Sandra

    2013-03-01

    The characterization of externally visible traits by DNA analysis is already an important tool when investigating ancient skeletal remains and may gain similar importance in future forensic DNA analysis. This, however, depends on the different legal regulations in the different countries. Besides eye or hair color, the population origin can provide crucial information in criminal prosecution. In this study, we present the analysis of 16 single-nucleotide polymorphisms (SNPs) combined to two robust SNaPshot assays with a detection threshold of 25-pg DNA. This assay was applied to 891 people from seven different populations (West Africa, North Africa, Turkey, Near East, Balkan states, North Europe, and Japan) with a thorough statistical evaluation. The prediction model was validated by an additional 125 individuals predominantly with an ancestry from those same regions. The specificity of these SNPs for the prediction of all population origins is very high (>90 %), but the sensitivity varied greatly (more than 90 % for West Africa, but only 25 % for the Near East). We could identify West Africans with a certainty of 100 %, and people from North Africa, the Balkan states, or North Europe nearly with the same reliability while determination of Turks or people from the Near East was rather difficult. In conclusion, the two SNaPshot assays are a powerful and reliable tool for the identification of people with an ancestry in one of the above listed populations, even from degraded DNA.

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

    PubMed

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

    2013-01-10

    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.

  12. Comprehensive comparative analysis of RNA sequencing methods for degraded or low input samples

    PubMed Central

    Adiconis, Xian; Borges-Rivera, Diego; Satija, Rahul; DeLuca, David S.; Busby, Michele A.; Berlin, Aaron M.; Sivachenko, Andrey; Thompson, Dawn Anne; Wysoker, Alec; Fennell, Timothy; Gnirke, Andreas; Pochet, Nathalie; Regev, Aviv; Levin, Joshua Z.

    2013-01-01

    RNA-Seq is an effective method to study the transcriptome, but can be difficult to apply to scarce or degraded RNA from fixed clinical samples, rare cell populations, or cadavers. Recent studies have proposed several methods for RNA-Seq of low quality and/or low quantity samples, but their relative merits have not been systematically analyzed. Here, we compare five such methods using metrics relevant to transcriptome annotation, transcript discovery, and gene expression. Using a single human RNA sample, we constructed and sequenced ten libraries with these methods and two control libraries. We find that the RNase H method performed best for low quality RNA, and confirmed this with actual degraded samples. RNase H can even effectively replace oligo (dT) based methods for standard RNA-Seq. SMART and NuGEN had distinct strengths for low quantity RNA. Our analysis allows biologists to select the most suitable methods and provides a benchmark for future method development. PMID:23685885

  13. Comparative analysis of RNA sequencing methods for degraded or low-input samples.

    PubMed

    Adiconis, Xian; Borges-Rivera, Diego; Satija, Rahul; DeLuca, David S; Busby, Michele A; Berlin, Aaron M; Sivachenko, Andrey; Thompson, Dawn Anne; Wysoker, Alec; Fennell, Timothy; Gnirke, Andreas; Pochet, Nathalie; Regev, Aviv; Levin, Joshua Z

    2013-07-01

    RNA-seq is an effective method for studying the transcriptome, but it can be difficult to apply to scarce or degraded RNA from fixed clinical samples, rare cell populations or cadavers. Recent studies have proposed several methods for RNA-seq of low-quality and/or low-quantity samples, but the relative merits of these methods have not been systematically analyzed. Here we compare five such methods using metrics relevant to transcriptome annotation, transcript discovery and gene expression. Using a single human RNA sample, we constructed and sequenced ten libraries with these methods and compared them against two control libraries. We found that the RNase H method performed best for chemically fragmented, low-quality RNA, and we confirmed this through analysis of actual degraded samples. RNase H can even effectively replace oligo(dT)-based methods for standard RNA-seq. SMART and NuGEN had distinct strengths for measuring low-quantity RNA. Our analysis allows biologists to select the most suitable methods and provides a benchmark for future method development.

  14. 77 FR 34387 - National Health and Nutrition Examination Survey (NHANES) DNA Samples

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-11

    ... Survey (NHANES) DNA Samples AGENCY: Centers for Disease Control and Prevention (CDC), Department of... 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 proposal guidelines. NHANES will announce when it...

  15. 76 FR 72417 - National Health and Nutrition Examination Survey (NHANES) DNA Samples

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-23

    ... National Health and Nutrition Examination Survey (NHANES) DNA Samples AGENCY: Centers for Disease Control... National Health and Nutrition 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 proposal...

  16. Degradable Polymer-Coated Gold Nanoparticles for Co-Delivery of DNA and siRNA

    PubMed Central

    Bishop, Corey J.; Tzeng, Stephany Y.; Green, Jordan J.

    2014-01-01

    Gold nanoparticles have utility for in vitro, ex vivo, and in vivo imaging applications as well as for serving as a scaffold for therapeutic delivery and theranostic applications. Starting with gold nanoparticles as a core, layer-by-layer degradable polymer coatings enable co-delivery of both DNA and short interfering RNA simultaneously. To engineer release kinetics, polymers which degrade through two different mechanisms can be utilized to construct hybrid inorganic/polymeric particles. During fabrication of the nanoparticles, the zeta potential reverses upon the addition of each oppositely charged polyelectrolyte layer and the final nanoparticle size reaches approximately 200 nm in diameter. When the hybrid gold/polymer/nucleic acid nanoparticles are added to human primary brain cancer cells in vitro, they are internalizable by cells and reach the cytoplasm and nucleus as visualized by transmission electron microscopy and observed through exogenous gene expression. This nanoparticle delivery leads to both exogenous DNA expression and siRNA-mediated knockdown, with the knockdown efficacy superior to that of Lipofectamine® 2000, a commercially available transfection reagent. These gold/polymer/nucleic acid hybrid nanoparticles are an enabling theranostic platform technology capable of delivering combinations of genetic therapies to human cells. PMID:25246314

  17. Degradable polymer-coated gold nanoparticles for co-delivery of DNA and siRNA.

    PubMed

    Bishop, Corey J; Tzeng, Stephany Y; Green, Jordan J

    2015-01-01

    Gold nanoparticles have utility for in vitro, ex vivo and in vivo imaging applications as well as for serving as a scaffold for therapeutic delivery and theranostic applications. Starting with gold nanoparticles as a core, layer-by-layer degradable polymer coatings enable the simultaneous co-delivery of DNA and short interfering RNA (siRNA). To engineer release kinetics, polymers which degrade through two different mechanisms can be utilized to construct hybrid inorganic/polymeric particles. During fabrication of the nanoparticles, the zeta potential reverses upon the addition of each oppositely charged polyelectrolyte layer and the final nanoparticle size reaches approximately 200nm in diameter. When the hybrid gold/polymer/nucleic acid nanoparticles are added to human primary brain cancer cells in vitro, they are internalizable by cells and reach the cytoplasm and nucleus as visualized by transmission electron microscopy and observed through exogenous gene expression. This nanoparticle delivery leads to both exogenous DNA expression and siRNA-mediated knockdown, with the knockdown efficacy superior to that of Lipofectamine® 2000, a commercially available transfection reagent. These gold/polymer/nucleic acid hybrid nanoparticles are an enabling theranostic platform technology capable of delivering combinations of genetic therapies to human cells.

  18. Comparison of seven methods for extraction of bacterial DNA from fecal and cecal samples of mice.

    PubMed

    Ferrand, Janina; Patron, Kevin; Legrand-Frossi, Christine; Frippiat, Jean-Pol; Merlin, Christophe; Alauzet, Corentine; Lozniewski, Alain

    2014-10-01

    Analysis of bacterial DNA from fecal samples of mice is commonly performed in experimental studies. Although DNA extraction is a critical step in various molecular approaches, the efficiency of methods that may be used for DNA extraction from mice fecal samples has never been evaluated. We compared the efficiencies of six widely used commercial kits (MasterPure™ Gram Positive DNA Purification Kit, QIAamp® DNA Stool Mini Kit; NucliSENS® easyMAG®, ZR Fecal DNA MiniPrep™, FastDNA® SPIN Kit for Feces and FastDNA® SPIN Kit for Soil) and a non-commercial method for DNA isolation from mice feces and cecal contents. DNA quantity and quality were assessed by fluorometry, spectrophotometry, gel electrophoresis and qPCR. Cell lysis efficiencies were evaluated by qPCR targeting three relevant bacteria in spiked specimens. For both feces and intestinal contents, the most efficient extraction method was the FastDNA® SPIN Kit for Soil.

  19. Avoiding Ethanol Presence in DNA Samples Enhances the Performance of Ultraviolet Resonance Raman Spectroscopy Analysis.

    PubMed

    Cammisuli, Francesca; Pascolo, Lorella; Morgutti, Marcello; Gessini, Alessandro; Masciovecchio, Claudio; D'Amico, Francesco

    2017-01-01

    Ethanol is an essential chemical reagent in DNA preparation as its use increases the yield of extraction. All methodologies for DNA isolation involve the use of ethanol in order to prevent DNA dissolution in water and to optimize the binding of DNA to chromatographic membranes. In this note, we show how the presence of ethanol traces in DNA aqueous solution affects ultraviolet Raman spectra, leading to possible misinterpretations. We report a simple method to remove the ethanol Raman features from the spectra, based on heating the DNA sample at 80 ℃, followed by a slow cooling procedure.

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

    PubMed

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

    2003-03-01

    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.

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

    PubMed

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

    2010-10-01

    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.

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

    PubMed Central

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

    2013-01-01

    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, γ-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. PMID:23847654

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

    ERIC Educational Resources Information Center

    Millard, Julie T

    2011-01-01

    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…

  4. Fluorescence formation from the interaction of DNA with lipid oxidation degradation products.

    PubMed

    Frankel, E N; Neff, W E; Brooks, D D; Fujimoto, K

    1987-06-23

    To clarify the mechanism of fluorescence formation between DNA and lipid degradation products in the presence of ferric chloride and ascorbic acid, a number of carbonyl compounds and decomposition products of pure methyl linolenate hydroperoxides were examined. Keto derivatives of methyl ricinoleate, linoleate, and oleate, alkanals and 2-alkenals produced little or no fluorescence with DNA in the presence of ferric chloride-ascorbic acid. 2,4-Alkadienals were more active and 2,4,7-decatrienal was the most active. Mixtures of volatile aldehydes prepared from linolenate hydroperoxide decomposed either thermally or with iron and ascorbate had the same activity as 2,4,7-decatrienal. Higher molecular-weight products from the decomposition of methyl linolenate hydroperoxides showed relatively low activity. beta-Carotene, alpha-tocopherol and other antioxidants effectively reduced the amount of fluorescence formed by linolenate hydroperoxides. The results suggest that, in addition to hydroperoxide decomposition products, singlet oxygen and/or free radical species contribute significantly to the fluorescence formed from the interaction of methyl linolenate hydroperoxides with DNA in the presence of ferric chloride and ascorbic acid.

  5. DNA extraction methods and multiple sampling to improve molecular diagnosis of Sarcocystis spp. in cattle hearts.

    PubMed

    Bräunig, Patrícia; Portella, Luiza Pires; Cezar, Alfredo Skrebsky; Libardoni, Felipe; Sangioni, Luis Antonio; Vogel, Fernanda Silveira Flores; Gonçalves, Paulo Bayard Dias

    2016-10-01

    Molecular detection of Sarcocystis spp. in tissue samples can be useful for experimental and diagnostic purposes. However, the parasite spreads unevenly through tissues, forming tissue cysts, and the cystic wall is an obstacle in DNA extraction protocols. Therefore, adequate sampling and effective disruption of the cysts are essential to improve the accuracy of DNA detection by PCR. The aims of this study were to evaluate the suitability of four protocols for DNA extraction from cysts of Sarcocystis spp. present in bovine myocardium samples or after their harvest in phosphate-buffered saline (PBS) solution as well as determine the effects of single or multiple sampling on the accuracy of molecular diagnosis of sarcocystosis in cattle hearts. Cysts and myocardium samples from nine bovine hearts were randomly distributed to four DNA extraction protocols: kit, kit with modification, DNAzol, and cetyl-trimethyl ammonium bromide (CTAB). Samples were submitted to DNA extraction and PCR as replicates of each heart (simplicate, duplicate, and triplicate), and the probability of a true positive diagnostic was calculated. Among the protocols tested, the kit with modification was determined to be the most suitable for DNA extraction from cysts in PBS solution (92.6 % of DNA detection by PCR); DNAzol resulted in higher DNA detection frequency from bovine myocardium samples (48.1 %). Multiple sampling improved the molecular diagnosis of Sarcocystis spp. infection in cattle hearts, increasing at 22.2 % the rate of true positive diagnostic.

  6. DNA from Dust: Comparative Genomics of Large DNA Viruses in Field Surveillance Samples.

    PubMed

    Pandey, Utsav; Bell, Andrew S; Renner, Daniel W; Kennedy, David A; Shreve, Jacob T; Cairns, Chris L; Jones, Matthew J; Dunn, Patricia A; Read, Andrew F; Szpara, Moriah L

    2016-01-01

    The intensification of the poultry industry over the last 60 years facilitated the evolution of increased virulence and vaccine breaks in Marek's disease virus (MDV-1). Full-genome sequences are essential for understanding why and how this evolution occurred, but what is known about genome-wide variation in MDV comes from laboratory culture. To rectify this, we developed methods for obtaining high-quality genome sequences directly from field samples without the need for sequence-based enrichment strategies prior to sequencing. We applied this to the first characterization of MDV-1 genomes from the field, without prior culture. These viruses were collected from vaccinated hosts that acquired naturally circulating field strains of MDV-1, in the absence of a disease outbreak. This reflects the current issue afflicting the poultry industry, where virulent field strains continue to circulate despite vaccination and can remain undetected due to the lack of overt disease symptoms. We found that viral genomes from adjacent field sites had high levels of overall DNA identity, and despite strong evidence of purifying selection, had coding variations in proteins associated with virulence and manipulation of host immunity. Our methods empower ecological field surveillance, make it possible to determine the basis of viral virulence and vaccine breaks, and can be used to obtain full genomes from clinical samples of other large DNA viruses, known and unknown. IMPORTANCE Despite both clinical and laboratory data that show increased virulence in field isolates of MDV-1 over the last half century, we do not yet understand the genetic basis of its pathogenicity. Our knowledge of genome-wide variation between strains of this virus comes exclusively from isolates that have been cultured in the laboratory. MDV-1 isolates tend to lose virulence during repeated cycles of replication in the laboratory, raising concerns about the ability of cultured isolates to accurately reflect virus in

  7. DNA from Dust: Comparative Genomics of Large DNA Viruses in Field Surveillance Samples

    PubMed Central

    Pandey, Utsav; Bell, Andrew S.; Renner, Daniel W.; Kennedy, David A.; Shreve, Jacob T.; Cairns, Chris L.; Jones, Matthew J.; Dunn, Patricia A.; Read, Andrew F.

    2016-01-01

    ABSTRACT The intensification of the poultry industry over the last 60 years facilitated the evolution of increased virulence and vaccine breaks in Marek’s disease virus (MDV-1). Full-genome sequences are essential for understanding why and how this evolution occurred, but what is known about genome-wide variation in MDV comes from laboratory culture. To rectify this, we developed methods for obtaining high-quality genome sequences directly from field samples without the need for sequence-based enrichment strategies prior to sequencing. We applied this to the first characterization of MDV-1 genomes from the field, without prior culture. These viruses were collected from vaccinated hosts that acquired naturally circulating field strains of MDV-1, in the absence of a disease outbreak. This reflects the current issue afflicting the poultry industry, where virulent field strains continue to circulate despite vaccination and can remain undetected due to the lack of overt disease symptoms. We found that viral genomes from adjacent field sites had high levels of overall DNA identity, and despite strong evidence of purifying selection, had coding variations in proteins associated with virulence and manipulation of host immunity. Our methods empower ecological field surveillance, make it possible to determine the basis of viral virulence and vaccine breaks, and can be used to obtain full genomes from clinical samples of other large DNA viruses, known and unknown. IMPORTANCE Despite both clinical and laboratory data that show increased virulence in field isolates of MDV-1 over the last half century, we do not yet understand the genetic basis of its pathogenicity. Our knowledge of genome-wide variation between strains of this virus comes exclusively from isolates that have been cultured in the laboratory. MDV-1 isolates tend to lose virulence during repeated cycles of replication in the laboratory, raising concerns about the ability of cultured isolates to accurately

  8. A bone sample cleaning method using trypsin for the isolation of DNA.

    PubMed

    Li, Richard; Liriano, Lidissy

    2011-11-01

    Cleaning the surface of bone samples is a necessary step to remove contaminants prior to isolating DNA for forensic DNA analysis. In this study, a simple trypsin method for cleaning bone samples prior to DNA isolation was developed. Cleaning the surface of human bone samples was achieved by the application of trypsin solution. Light microscopy and scanning electron microscopy results indicated that trypsin treatment was effective in removing the outer surface of bone samples. The yield of DNA isolated from trypsin-treated bone samples was sufficient for subsequent short tandem repeat (STR) analysis. STR analysis revealed no adverse effect on the DNA profile after the trypsin treatment. The data suggest that this trypsin method can potentially be an alternative cleaning method to mechanical cleaning methods.

  9. 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

    Eckels, David E.; Hass, William J.

    1989-05-30

    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.

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

    USGS Publications Warehouse

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

    2013-01-01

    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.

  11. Stabilization of human urine doping control samples: II. microbial degradation of steroids.

    PubMed

    Tsivou, M; Livadara, D; Georgakopoulos, D G; Koupparis, M A; Atta-Politou, J; Georgakopoulos, C G

    2009-05-01

    The transportation of urine samples, collected for doping control analysis, does not always meet ideal conditions of storage and prompt delivery to the World Anti-Doping Agency (WADA) accredited laboratories. Because sample collection is not conducted under sterile conditions, microbial activity may cause changes to the endogenous steroid profiles of samples. In the current work, funded by WADA, a chemical mixture consisting of antibiotics, antimycotic substances and protease inhibitors was applied in urine aliquots fortified with conjugated and deuterated steroids and inoculated with nine representative microorganisms. Aliquots with and without the chemical mixture were incubated at 37 degrees C for 7 days to simulate the transportation period, whereas another series of aliquots was stored at -20 degrees C as reference. Microbial growth was assessed immediately after inoculation and at the end of the incubation period. Variations in pH and specific gravity values were recorded. Gas chromatography-mass spectrometry (GC-MS) analysis was performed for the detection of steroids in the free, glucuronide, and sulfate fractions. The addition of the chemical stabilization mixture to urine samples inhibited microorganism growth and prevented steroid degradation at 37 degrees C. On the other hand, four of the nine microorganisms induced alterations in the steroid profile of the unstabilized samples incubated at 37 degrees C.

  12. Computational analyses of ancient pathogen DNA from herbarium samples: challenges and prospects

    PubMed Central

    Yoshida, Kentaro; Sasaki, Eriko; Kamoun, Sophien

    2015-01-01

    The application of DNA sequencing technology to the study of ancient DNA has enabled the reconstruction of past epidemics from genomes of historically important plant-associated microbes. Recently, the genome sequences of the potato late blight pathogen Phytophthora infestans were analyzed from 19th century herbarium specimens. These herbarium samples originated from infected potatoes collected during and after the Irish potato famine. Herbaria have therefore great potential to help elucidate past epidemics of crops, date the emergence of pathogens, and inform about past pathogen population dynamics. DNA preservation in herbarium samples was unexpectedly good, raising the possibility of a whole new research area in plant and microbial genomics. However, the recovered DNA can be extremely fragmented resulting in specific challenges in reconstructing genome sequences. Here we review some of the challenges in computational analyses of ancient DNA from herbarium samples. We also applied the recently developed linkage method to haplotype reconstruction of diploid or polyploid genomes from fragmented ancient DNA. PMID:26442080

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

    PubMed

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

    2008-08-01

    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.

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

    NASA Astrophysics Data System (ADS)

    Millard, Julie T.; Pilon, André M.

    2003-04-01

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

  15. Degradation of paternal mitochondria after fertilization: implications for heteroplasmy, assisted reproductive technologies and mtDNA inheritance.

    PubMed

    Sutovsky, Peter; Van Leyen, Klaus; McCauley, Tod; Day, Billy N; Sutovsky, Miriam

    2004-01-01

    Maternal inheritance of mitochondrial DNA has long been regarded as a major paradox in developmental biology. While some confusion may still persist in popular science, research data clearly document that the paternal sperm-borne mitochondria of most mammalian species enter the ooplasm at fertilization and are specifically targeted for degradation by the resident ubiquitin system. Ubiquitin is a proteolytic chaperone that forms covalently linked polyubiquitin chains on the targeted proteinaceous substrates. The polyubiquitin tag redirects the substrate proteins to a 26-S proteasome, a multi-subunit proteolytic organelle. Thus, specific proteasomal inhibitors reversibly block sperm mitochondrial degradation in ooplasm. Lysosomal degradation and the activity of membrane-lipoperoxidating enzyme 15-lipoxygenase (15-LOX) may also contribute to sperm mitochondrial degradation in the ooplasm, but probably is not crucial. Prohibitin, the major protein of the inner mitochondrial membrane, appears to be ubiquitinated in the sperm mitochondria. Occasional occurrence of paternal inheritance of mtDNA has been suggested in mammals including humans. While most such evidence has been widely disputed, it warrants further examination. Of particular concern is the documented heteroplasmy, i.e. mixed mtDNA inheritance after ooplasmic transplantation. Intracytoplasmic sperm injection (ICSI) has inherent potential for delaying the degradation of sperm mitochondria. However, paternal mtDNA inheritance after ICSI has not been documented so far.

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

    PubMed

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

    2013-08-01

    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.

  17. Storage and shipping of tissue samples for DNA analyses: A case study on earthworms☆

    PubMed Central

    Straube, Daniela; Juen, Anita

    2013-01-01

    Nowadays, molecular analyses play an important role in studies of soil dwelling animals, for example in taxonomy, phylogeography or food web analyses. The quality of the DNA, used for later molecular analyses, is an important factor and depends on collection and preservation of samples prior to DNA extraction. Ideally, DNA samples are frozen immediately upon collection, but if samples are collected in the field, suitable preservation methods might be limited due to unavailability of resources or remote field sites. Moreover, shipping samples over long distances can cause loss of DNA quality e.g. by thawing or leaking of preservation liquid. In this study we use earthworms, a key organism in soil research, to compare three different DNA preservation methods – freezing at −20 °C, storing in 75% ethanol, and freeze drying. Samples were shipped from the United States of America to Austria. The DNA of the samples was extracted using two different extraction methods, peqGOLD™ and Chelex® 100. The DNA amplification success was determined by amplifying four DNA fragments of different length. The PCR amplification success is significantly influenced by preservation method and extraction method and differed significantly depending on the length of the DNA fragment. Freeze drying samples was the best preservation method when samples were extracted using the silica based extraction method peqGOLD™. For samples that were extracted with Chelex® 100, storage in ethanol was the best preservation method. However, the overall amplification success was significantly lower for the extraction procedure based on Chelex® 100. The detection of the small DNA fragments was higher and independent from the extraction method, while the amplification success was significantly reduced for the longer DNA fragments. We recommend freeze drying of DNA samples, especially when they have to be shipped for longer distances. No special packaging or declaration is needed for freeze dried

  18. Wild chimpanzee infant urine and saliva sampled noninvasively usable for DNA analyses.

    PubMed

    Inoue, Eiji; Inoue-Murayama, Miho; Takenaka, Osamu; Nishida, Toshisada

    2007-04-01

    In many genetic studies on the great apes, fecal or hair samples have been used as sources of DNA. However, feces and hairs are difficult to collect from chimpanzee infants under 3 years of age. As alternative DNA sources, we investigated the efficiency of collecting urine samples from infants compared with fecal samples, as well as the validity of the DNA extracted from urine and saliva samples of well-habituated M group chimpanzees (Pan troglodytes schweinfurthii) in the Mahale Mountains National Park, Tanzania. We collected 40 urine and 3 fecal samples from 10 infants under 3 years. Compared with feces, the urine samples were relatively easy to collect. The saliva of infants, which remained on the twigs sucked by them, was collected using cotton swabs. The average amounts of DNA extracted from the 40 urine and 6 saliva samples were 3,920 and 458 pg/mul, respectively. The rate of positive PCR was low and the allelic dropout rate was high when using less than 25 pg of template DNA in the PCR mixtures. Based on the amounts of DNA, 50% of the urine samples and 100% of the saliva samples were judged usable for accurate microsatellite genotyping. For infant chimpanzees in particular, collecting urine and saliva as an alternative to fecal and hair samples can reduce the effort invested in collection in the field.

  19. Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment

    PubMed Central

    Song, Chen; Liu, Yibin; Fontana, Rachel; Makrigiorgos, Alexander; Mamon, Harvey; Kulke, Matthew H.; Makrigiorgos, G. Mike

    2016-01-01

    Presence of excess unaltered, wild-type (WT) DNA providing no information of biological or clinical value often masks rare alterations containing diagnostic or therapeutic clues in cancer, prenatal diagnosis, infectious diseases or organ transplantation. With the surge of high-throughput technologies there is a growing demand for removing unaltered DNA over large pools-of-sequences. Here we present nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO), a single-step approach with broad genome coverage that can remove WT-DNA from numerous sequences simultaneously, prior to genomic analysis. NaME-PrO employs a double-strand-DNA-specific nuclease and overlapping oligonucleotide-probes interrogating WT-DNA targets and guiding nuclease digestion to these sites. Mutation-containing DNA creates probe-DNA mismatches that inhibit digestion, thus subsequent DNA-amplification magnifies DNA-alterations at all selected targets. We demonstrate several-hundred-fold mutation enrichment in diverse human samples on multiple clinically relevant targets including tumor samples and circulating DNA in 50-plex reactions. Enrichment enables routine mutation detection at 0.01% abundance while by adjusting conditions it is possible to sequence mutations down to 0.00003% abundance, or to scan tumor-suppressor genes for rare mutations. NaME-PrO introduces a simple and highly parallel process to remove un-informative DNA sequences and unmask clinically and biologically useful alterations. PMID:27431322

  20. PNA microarrays for hybridisation of unlabelled DNA samples

    PubMed Central

    Brandt, Ole; Feldner, Julia; Stephan, Achim; Schröder, Markus; Schnölzer, Martina; Arlinghaus, Heinrich F.; Hoheisel, Jörg D.; Jacob, Anette

    2003-01-01

    Several strategies have been developed for the production 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 from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which had been chemically introduced during synthesis. Thus, only full-length PNA oligomers were attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, did not bind. Different surface chemistries and fitting modifications of the PNA terminus were tested. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI-TOF mass spectrometry. Additionally, hybridisation experiments were performed to compare the attachment chemistries, with fully acetylated PNAs spotted as controls. Upon hybridisation of unlabelled DNA to such microarrays, binding events could be detected by visualisation of phosphates, which are an integral part of nucleic acids but missing entirely in PNA probes. Overall best results in terms of selectivity and sensitivity were obtained with thiol-modified PNAs on maleimide surfaces. PMID:14500847

  1. The influence of swabbing solutions on DNA recovery from touch samples.

    PubMed

    Thomasma, Sarah M; Foran, David R

    2013-03-01

    There has been minimal research into how to best obtain DNA from touch samples. Many forensic laboratories simply moisten a swab with water and use it for collecting cells/DNA from evidentiary samples. However, this and other methods have not been objectively studied in order to maximize DNA yields. In this study, fingerprints were collected using swabs moistened with water or laboratory or commercially available detergents, including sodium dodecyl sulfate (SDS), Triton X-100, Tween 20, Formula 409(®) , and Simple Green(®) . Prints were swabbed, DNA isolated using an organic extraction, yields quantified, and relative yields compared. In all cases, the detergent-based swabbing solutions outperformed water, with SDS and Triton X-100 producing significant increases in yield. Short tandem repeat profiles were consistent with the individuals that placed them. Subsequent analysis of SDS concentrations for collecting touch DNA demonstrated an increase in DNA yield with increasing SDS concentration, with an optimal concentration of approximately 2%.

  2. A comparative evaluation of four DNA extraction protocols from whole blood sample.

    PubMed

    Ghaheri, M; Kahrizi, D; Yari, K; Babaie, A; Suthar, R S; Kazemi, E

    2016-03-31

    All organisms have Deoxyribonucleic acid (DNA) within their cells. DNA is a complex molecule that contains all of the information necessary to build and maintain an organism. DNA extraction is one of the most basic and essential techniques in the study of DNA that allow huge advances in molecular biology, biotechnology and bioinformatics laboratories. Whole blood samples are one of the main sources used to obtain DNA and there are many different protocols available in this issue. In current research, compared four DNA extraction protocols from blood samples; include modified phenol-chloroform protocol, two salting-out and enzyme free method and from commercial kit. The extracted DNAs by these protocols were analyzed according to their time demands, quality and quantity, toxicity and functionality in PCR method. Also the quality and quantity of the extracted DNA were surveyed by gel electrophoresis and Nanodrop spectrophotometry methods. It was observed that there are not significantly differences between these methods about DNA Purity (A260/A280), but the DNA yield (ng DNA/μl) of phenol/chloroform method was higher than other methods. In addition, phenol/chloroform was the most toxic method and it takes more time than other methods. Roche diagnostics GmbH kit was the most expensive among the four methods but the least extraction time was required and it was the safest method.

  3. High-throughput DNA isolation method for detection of Xylella fastidiosa in plant and insect samples.

    PubMed

    Brady, Jeff A; Faske, Jennifer B; Castañeda-Gill, Jessica M; King, Jonathan L; Mitchell, Forrest L

    2011-09-01

    We report an inexpensive, high-throughput method for isolating DNA from insect and plant samples for the purpose of detecting Xylella fastidiosa infection. Existing methods often copurify inhibitors of DNA polymerases, limiting their usefulness for PCR-based detection assays. When compared to commercially available kits, the method provides enhanced pathogen detection at a fraction of the cost.

  4. Antisera specific for carcinogen-DNA adducts and carcinogen-modified DNA: applications for detection of xenobiotics in biological samples.

    PubMed

    Poirier, M C

    1993-07-01

    The development of immunoassays and immunoaffinity chromatography methods for determination of carcinogen-DNA adducts and carcinogen-modified DNA samples rests upon eliciting and characterizing polyclonal and monoclonal antisera against these haptens. The use of such antisera has widespread application in investigating chronic carcinogen administration in animal models and in monitoring human tissues for evidence of carcinogen exposure. Radioimmunoassays and enzyme-linked immunosorbent assays developed with carcinogen-DNA adduct antisera are exceedingly sensitive, measuring 1 adduct in 10(8) nucleotides. Not only can DNA damage be quantified directly by immunoassay, but the antisera have also been used to isolate DNA adducts of a particular chemical class by immunoaffinity chromatography before application of more chemically-specific end-points. Both of these methodological approaches have made seminal contributions to the newly-emerging field of molecular epidemiology. This chapter will focus on methods for preparing immunogens, the establishment of immunoassays, characterization of antisera and specific problems encountered with biological samples in addition, the use of immunoaffinity chromatography for preparative concentration of DNA adducts of a particular class will be included.

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

    PubMed

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

    2014-01-01

    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.

  6. Developing a simple method to process bone samples prior to DNA isolation.

    PubMed

    Li, Richard; Chapman, Sandra; Thompson, Mary; Schwartz, Michal

    2009-03-01

    Bone tissue is often used for recovering DNA samples for the purpose of human identification. However, the initial cleaning and sampling of the bone specimen is a labor-intensive and time-consuming step, which must be completed prior to isolating DNA. Thus, it is difficult to adapt the current method for automation. To address this issue, we have developed a simple processing method using a trypsin treatment prior to DNA isolation. The use of the trypsin-based procedure potentially reduces the amount of labor required by a physical method such as sanding. By incubating samples with the trypsin solution, the soft tissue and outer surface of the bone fragment samples are removed. The processed bone fragment or a portion of the fragment can then be used for DNA isolation.

  7. Comparing different post-mortem human samples as DNA sources for downstream genotyping and identification.

    PubMed

    Calacal, Gayvelline C; Apaga, Dame Loveliness T; Salvador, Jazelyn M; Jimenez, Joseph Andrew D; Lagat, Ludivino J; Villacorta, Renato Pio F; Lim, Maria Cecilia F; Fortun, Raquel D R; Datar, Francisco A; De Ungria, Maria Corazon A

    2015-11-01

    The capability of DNA laboratories to perform genotyping procedures from post-mortem remains, including those that had undergone putrefaction, continues to be a challenge in the Philippines, a country characterized by very humid and warm conditions all year round. These environmental conditions accelerate the decomposition of human remains that were recovered after a disaster and those that were left abandoned after a crime. When considerable tissue decomposition of human remains has taken place, there is no other option but to extract DNA from bone and/or teeth samples. Routinely, femur shafts are obtained from recovered bodies for human identification because the calcium matrix protects the DNA contained in the osteocytes. In the Philippines, there is difficulty in collecting femur samples after natural disasters or even human-made disasters, because these events are usually characterized by a large number of fatalities. Identification of casualties is further delayed by limitation in human and material resources. Hence, it is imperative to test other types of biological samples that are easier to collect, transport, process and store. We analyzed DNA that were obtained from body fluid, bone marrow, muscle tissue, clavicle, femur, metatarsal, patella, rib and vertebral samples from five recently deceased untreated male cadavers and seven male human remains that were embalmed, buried for ∼ 1 month and then exhumed. The bodies had undergone different environmental conditions and were in various stages of putrefaction. A DNA extraction method utilizing a detergent-washing step followed by an organic procedure was used. The utility of bone marrow and vitreous fluid including bone marrow and vitreous fluid that was transferred on FTA(®) cards and subjected to autosomal STR and Y-STR DNA typing were also evaluated. DNA yield was measured and the presence or absence of PCR inhibitors in DNA extracts was assessed using Plexor(®)HY. All samples were amplified using

  8. A procedure for separate recovery of extra- and intracellular DNA from a single marine sediment sample.

    PubMed

    Alawi, Mashal; Schneider, Beate; Kallmeyer, Jens

    2014-09-01

    Extracellular DNA (eDNA) is a ubiquitous biological compound in aquatic sediment and soil. Previous studies suggested that eDNA plays an important role in biogeochemical element cycling, horizontal gene transfer and stabilization of biofilm structures. Previous methods for eDNA extraction were either not suitable for oligotrophic sediments or only allowed quantification but no genetic analyses. Our procedure is based on cell detachment and eDNA liberation from sediment particles by sequential washing with an alkaline sodium phosphate buffer followed by a separation of cells and eDNA. The separated eDNA is then bound onto silica particles and purified, whereas the intracellular DNA from the separated cells is extracted using a commercial kit. The method provides extra- and intracellular DNA of high purity that is suitable for downstream applications like PCR. Extracellular DNA was extracted from organic-rich shallow sediment of the Baltic Sea, glacially influenced sediment of the Barents Sea and from the oligotrophic South Pacific Gyre. The eDNA concentration in these samples varied from 23 to 626ngg(-1) wet weight sediment. A number of experiments were performed to verify each processing step. Although extraction efficiency is higher than other published methods, it is not fully quantitative.

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

    PubMed

    Guo, Feng; Zhang, Tong

    2013-05-01

    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.

  10. Hydroxyl-radical-dependent DNA damage by ambient particulate matter from contrasting sampling locations

    SciTech Connect

    Shi Tingming; Duffin, Rodger; Borm, Paul J.A.; Li Hui; Weishaupt, Christel; Schins, Roel P.F. . E-mail: roel.schins@uni-duesseldorf.de

    2006-05-15

    Exposure to ambient particulate matter (PM) has been reported to be associated with increased respiratory, cardiovascular, and malignant lung disease. Previously we have shown that PM can induce oxidative DNA damage in A549 human lung epithelial cells. The aims of the present study were to investigate the variability of the DNA-damaging properties of PM sampled at different locations and times and to relate the observed effects to the hydroxyl-radical ({center_dot}OH)-generating activities of these samples. Weekly samples of coarse (10-2.5 {mu}m) and fine (<2.5 {mu}m) PM from four sites (Nordrheim Westfalen, Germany) were analyzed for hydrogen-peroxide-dependent {center_dot}OH formation using electron paramagnetic resonance and formation of 8-hydroxydeoxyguanosine (8-OHdG) in calf thymus DNA using an immuno-dot-blot assay. DNA strand breakage by fine PM in A549 human lung epithelial cells was quantified using the alkaline comet assay. Both PM size distribution fractions elicited {center_dot}OH generation and 8-OHdG formations in calf thymus DNA. Significantly higher {center_dot}OH generation was observed for PM sampled at urban/industrial locations and for coarse PM. Samples of fine PM also caused DNA strand breakage in A549 cells and this damage could be prevented using the hydroxyl-radical scavengers 5,5-dimethyl-1-pyrroline-N-oxide and dimethyl sulfoxide. The observed DNA strand breakage appeared to correlate with the hydroxyl-radical-generating capacities of the PM samples but with different profiles for rural versus urban/industrial samples. In conclusion, when considered at equal mass, {center_dot}OH formation of PM shows considerable variability with regard to the sampling location and time and is correlated with its ability to cause DNA damage.

  11. Enumeration of polysaccharide-degrading Bacteroides species in human feces by using species-specific DNA probes.

    PubMed Central

    Kuritza, A P; Shaughnessy, P; Salyers, A A

    1986-01-01

    DNA probes that are specific for each of five predominant species of human colonic Bacteroides (B. thetaiotaomicron, B. uniformis, B. distasonis, "Bacteroides group 3452-A", and B. ovatus) were used to detect and enumerate these species in fecal samples from two adult volunteers. These five species are capable of fermenting many of the complex polysaccharides that are thought to be sources of carbon and energy for bacteria in the colon. Estimates for the concentrations of some of these species in feces have not been previously available because of the difficulties in differentiating colonic Bacteroides spp. by conventional biochemical tests. Our results indicate that all the species except B. ovatus were present in high numbers (greater than 10(9)/g [dry weight]) in the feces of both volunteers. However, the concentrations of the more versatile polysaccharide-degrading species within this group of organisms (7.6 X 10(9) to 12.0 X 10(9)/g [dry weight] for B. thetaiotaomicron; 2.9 X 10(9) to 6.3 X 10(9)/g [dry weight] for "Bacteroides group 3452-A") did not differ significantly from the concentrations of less versatile polysaccharide-degrading species (1.2 X 10(10) to 2.0 X 10(10)/g [dry weight] for B. uniformis; 5.8 X 10(9) to 8.4 X 10(9)/g [dry weight] for B. distasonis). B. ovatus was not detectable by our method. Since our lower limit of detection is approximately 1 X 10(9) to 2 X 10(9)/g (dry weight) of feces, this is consistent with earlier estimates that indicated that the concentration of B. ovatus in feces is near or below this value.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3954350

  12. Enumeration of polysaccharide-degrading Bacteroides species in human feces by using species-specific DNA probes.

    PubMed

    Kuritza, A P; Shaughnessy, P; Salyers, A A

    1986-02-01

    DNA probes that are specific for each of five predominant species of human colonic Bacteroides (B. thetaiotaomicron, B. uniformis, B. distasonis, "Bacteroides group 3452-A", and B. ovatus) were used to detect and enumerate these species in fecal samples from two adult volunteers. These five species are capable of fermenting many of the complex polysaccharides that are thought to be sources of carbon and energy for bacteria in the colon. Estimates for the concentrations of some of these species in feces have not been previously available because of the difficulties in differentiating colonic Bacteroides spp. by conventional biochemical tests. Our results indicate that all the species except B. ovatus were present in high numbers (greater than 10(9)/g [dry weight]) in the feces of both volunteers. However, the concentrations of the more versatile polysaccharide-degrading species within this group of organisms (7.6 X 10(9) to 12.0 X 10(9)/g [dry weight] for B. thetaiotaomicron; 2.9 X 10(9) to 6.3 X 10(9)/g [dry weight] for "Bacteroides group 3452-A") did not differ significantly from the concentrations of less versatile polysaccharide-degrading species (1.2 X 10(10) to 2.0 X 10(10)/g [dry weight] for B. uniformis; 5.8 X 10(9) to 8.4 X 10(9)/g [dry weight] for B. distasonis). B. ovatus was not detectable by our method. Since our lower limit of detection is approximately 1 X 10(9) to 2 X 10(9)/g (dry weight) of feces, this is consistent with earlier estimates that indicated that the concentration of B. ovatus in feces is near or below this value.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Sensitive DIP-STR markers for the analysis of unbalanced mixtures from "touch" DNA samples.

    PubMed

    Oldoni, Fabio; Castella, Vincent; Grosjean, Frederic; Hall, Diana

    2017-02-14

    Casework samples collected for forensic DNA analysis can produce genomic mixtures in which the DNA of the alleged offender is masked by high quantities of DNA coming from the victim. DIP-STRs are novel genetic markers specifically developed to enable the target analysis of a DNA of interest in the presence of exceeding quantities of a second DNA (up to 1000-fold). The genotyping system, which is based on allele-specific amplifications of haplotypes formed by a deletion/insertion polymorphism (DIP) and a short tandem repeat (STR), combines the capacity of targeting the DNA of an individual with a strong identification power. Finally, DIP-STRs are autosomal markers therefore they can be applied to any combination of major and minor DNA. In this study we aimed to assess the ability of DIP-STRs to detect the minor contributor on challenging "touch" DNA samples simulated with representative crime-associated substrates and to compare their performance to commonly used male-specific markers (Y-STRs). As part of a comprehensive study on the relative DNA contribution of two persons handling the same object, we selected 71 unbalanced contact traces of which 14 comprised a male minor DNA contributor mixed to a female major DNA contributor. Using a set of six DIP-STRs, one to four markers were found to be informative for the minor DNA detection across traces. When compared to Y-STRs (14 traces), the DIP-STRs showed similar sensitivity in detecting the minor DNA across substrate materials with a similar occurrence of allele drop-out. Conversely, because of the sex combination of the two users of the object, 57 remaining traces could only be investigated by DIP-STRs. Of these, 30 minor DNA contributors could be detected by all informative markers while 12 traces showed events of allele drop-out. Finally, 15 traces showed no amplification of the minor DNA. These last 15 samples were mostly characterized by a combination of short handling time of the object, low DNA recovery and

  14. Comparative analysis of metagenomes from three methanogenic hydrocarbon-degrading enrichment cultures with 41 environmental samples

    PubMed Central

    Tan, Boonfei; Jane Fowler, S; Laban, Nidal Abu; Dong, Xiaoli; Sensen, Christoph W; Foght, Julia; Gieg, Lisa M

    2015-01-01

    Methanogenic hydrocarbon metabolism is a key process in subsurface oil reservoirs and hydrocarbon-contaminated environments and thus warrants greater understanding to improve current technologies for fossil fuel extraction and bioremediation. In this study, three hydrocarbon-degrading methanogenic cultures established from two geographically distinct environments and incubated with different hydrocarbon substrates (added as single hydrocarbons or as mixtures) were subjected to metagenomic and 16S rRNA gene pyrosequencing to test whether these differences affect the genetic potential and composition of the communities. Enrichment of different putative hydrocarbon-degrading bacteria in each culture appeared to be substrate dependent, though all cultures contained both acetate- and H2-utilizing methanogens. Despite differing hydrocarbon substrates and inoculum sources, all three cultures harbored genes for hydrocarbon activation by fumarate addition (bssA, assA, nmsA) and carboxylation (abcA, ancA), along with those for associated downstream pathways (bbs, bcr, bam), though the cultures incubated with hydrocarbon mixtures contained a broader diversity of fumarate addition genes. A comparative metagenomic analysis of the three cultures showed that they were functionally redundant despite their enrichment backgrounds, sharing multiple features associated with syntrophic hydrocarbon conversion to methane. In addition, a comparative analysis of the culture metagenomes with those of 41 environmental samples (containing varying proportions of methanogens) showed that the three cultures were functionally most similar to each other but distinct from other environments, including hydrocarbon-impacted environments (for example, oil sands tailings ponds and oil-affected marine sediments). This study provides a basis for understanding key functions and environmental selection in methanogenic hydrocarbon-associated communities. PMID:25734684

  15. Comparative analysis of metagenomes from three methanogenic hydrocarbon-degrading enrichment cultures with 41 environmental samples.

    PubMed

    Tan, Boonfei; Fowler, S Jane; Abu Laban, Nidal; Dong, Xiaoli; Sensen, Christoph W; Foght, Julia; Gieg, Lisa M

    2015-09-01

    Methanogenic hydrocarbon metabolism is a key process in subsurface oil reservoirs and hydrocarbon-contaminated environments and thus warrants greater understanding to improve current technologies for fossil fuel extraction and bioremediation. In this study, three hydrocarbon-degrading methanogenic cultures established from two geographically distinct environments and incubated with different hydrocarbon substrates (added as single hydrocarbons or as mixtures) were subjected to metagenomic and 16S rRNA gene pyrosequencing to test whether these differences affect the genetic potential and composition of the communities. Enrichment of different putative hydrocarbon-degrading bacteria in each culture appeared to be substrate dependent, though all cultures contained both acetate- and H2-utilizing methanogens. Despite differing hydrocarbon substrates and inoculum sources, all three cultures harbored genes for hydrocarbon activation by fumarate addition (bssA, assA, nmsA) and carboxylation (abcA, ancA), along with those for associated downstream pathways (bbs, bcr, bam), though the cultures incubated with hydrocarbon mixtures contained a broader diversity of fumarate addition genes. A comparative metagenomic analysis of the three cultures showed that they were functionally redundant despite their enrichment backgrounds, sharing multiple features associated with syntrophic hydrocarbon conversion to methane. In addition, a comparative analysis of the culture metagenomes with those of 41 environmental samples (containing varying proportions of methanogens) showed that the three cultures were functionally most similar to each other but distinct from other environments, including hydrocarbon-impacted environments (for example, oil sands tailings ponds and oil-affected marine sediments). This study provides a basis for understanding key functions and environmental selection in methanogenic hydrocarbon-associated communities.

  16. Structural insights into apoptotic DNA degradation by CED-3 protease suppressor-6 (CPS-6) from Caenorhabditis elegans.

    PubMed

    Lin, Jason L J; Nakagawa, Akihisa; Lin, Chia Liang; Hsiao, Yu-Yuan; Yang, Wei-Zen; Wang, Yi-Ting; Doudeva, Lyudmila G; Skeen-Gaar, Riley Robert; Xue, Ding; Yuan, Hanna S

    2012-03-02

    Endonuclease G (EndoG) is a mitochondrial protein that traverses to the nucleus and participates in chromosomal DNA degradation during apoptosis in yeast, worms, flies, and mammals. However, it remains unclear how EndoG binds and digests DNA. Here we show that the Caenorhabditis elegans CPS-6, a homolog of EndoG, is a homodimeric Mg(2+)-dependent nuclease, binding preferentially to G-tract DNA in the optimum low salt buffer at pH 7. The crystal structure of CPS-6 was determined at 1.8 Å resolution, revealing a mixed αβ topology with the two ββα-metal finger nuclease motifs located distantly at the two sides of the dimeric enzyme. A structural model of the CPS-6-DNA complex suggested a positively charged DNA-binding groove near the Mg(2+)-bound active site. Mutations of four aromatic and basic residues: Phe(122), Arg(146), Arg(156), and Phe(166), in the protein-DNA interface significantly reduced the DNA binding and cleavage activity of CPS-6, confirming that these residues are critical for CPS-6-DNA interactions. In vivo transformation rescue experiments further showed that the reduced DNase activity of CPS-6 mutants was positively correlated with its diminished cell killing activity in C. elegans. Taken together, these biochemical, structural, mutagenesis, and in vivo data reveal a molecular basis of how CPS-6 binds and hydrolyzes DNA to promote cell death.

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

    PubMed

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

    2014-05-01

    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.

  18. A Microbiome DNA Enrichment Method for Next-Generation Sequencing Sample Preparation.

    PubMed

    Yigit, Erbay; Feehery, George R; Langhorst, Bradley W; Stewart, Fiona J; Dimalanta, Eileen T; Pradhan, Sriharsa; Slatko, Barton; Gardner, Andrew F; McFarland, James; Sumner, Christine; Davis, Theodore B

    2016-07-01

    "Microbiome" is used to describe the communities of microorganisms and their genes in a particular environment, including communities in association with a eukaryotic host or part of a host. One challenge in microbiome analysis concerns the presence of host DNA in samples. Removal of host DNA before sequencing results in greater sequence depth of the intended microbiome target population. This unit describes a novel method of microbial DNA enrichment in which methylated host DNA such as human genomic DNA is selectively bound and separated from microbial DNA before next-generation sequencing (NGS) library construction. This microbiome enrichment technique yields a higher fraction of microbial sequencing reads and improved read quality resulting in a reduced cost of downstream data generation and analysis. © 2016 by John Wiley & Sons, Inc.

  19. Automated Device for Asynchronous Extraction of RNA, DNA, or Protein Biomarkers from Surrogate Patient Samples.

    PubMed

    Bitting, Anna L; Bordelon, Hali; Baglia, Mark L; Davis, Keersten M; Creecy, Amy E; Short, Philip A; Albert, Laura E; Karhade, Aditya V; Wright, David W; Haselton, Frederick R; Adams, Nicholas M

    2016-12-01

    Many biomarker-based diagnostic methods are inhibited by nontarget molecules in patient samples, necessitating biomarker extraction before detection. We have developed a simple device that purifies RNA, DNA, or protein biomarkers from complex biological samples without robotics or fluid pumping. The device design is based on functionalized magnetic beads, which capture biomarkers and remove background biomolecules by magnetically transferring the beads through processing solutions arrayed within small-diameter tubing. The process was automated by wrapping the tubing around a disc-like cassette and rotating it past a magnet using a programmable motor. This device recovered biomarkers at ~80% of the operator-dependent extraction method published previously. The device was validated by extracting biomarkers from a panel of surrogate patient samples containing clinically relevant concentrations of (1) influenza A RNA in nasal swabs, (2) Escherichia coli DNA in urine, (3) Mycobacterium tuberculosis DNA in sputum, and (4) Plasmodium falciparum protein and DNA in blood. The device successfully extracted each biomarker type from samples representing low levels of clinically relevant infectivity (i.e., 7.3 copies/µL of influenza A RNA, 405 copies/µL of E. coli DNA, 0.22 copies/µL of TB DNA, 167 copies/µL of malaria parasite DNA, and 2.7 pM of malaria parasite protein).

  20. Assessment of methods to recover DNA from bacteria, fungi and archaea in complex environmental samples.

    PubMed

    Guillén-Navarro, Karina; Herrera-López, David; López-Chávez, Mariana Y; Cancino-Gómez, Máximo; Reyes-Reyes, Ana L

    2015-11-01

    DNA extraction from environmental samples is a critical step for metagenomic analysis to study microbial communities, including those considered uncultivable. Nevertheless, obtaining good quality DNA in sufficient quantities for downstream methodologies is not always possible, and it depends on the complexity and stability of each ecosystem, which could be more problematic for samples from tropical regions because those ecosystems are less stable and more complex. Three laboratory methods for the extraction of nucleic acids from samples representing unstable (decaying coffee pulp and mangrove sediments) and relatively stable (compost and soil) environments were tested. The results were compared with those obtained using two commercial DNA extraction kits. The quality of the extracted DNA was evaluated by PCR amplification to verify the recovery of bacterial, archaeal, and fungal genetic material. The laboratory method that gave the best results used a lysis procedure combining physical, chemical, and enzymatic steps.

  1. Inspecting close maternal relatedness: Towards better mtDNA population samples in forensic databases

    PubMed Central

    Bodner, Martin; Irwin, Jodi A.; Coble, Michael D.; Parson, Walther

    2011-01-01

    Reliable data are crucial for all research fields applying mitochondrial DNA (mtDNA) as a genetic marker. Quality control measures have been introduced to ensure the highest standards in sequence data generation, validation and a posteriori inspection. A phylogenetic alignment strategy has been widely accepted as a prerequisite for data comparability and database searches, for forensic applications, for reconstructions of human migrations and for correct interpretation of mtDNA mutations in medical genetics. There is continuing effort to enhance the number of worldwide population samples in order to contribute to a better understanding of human mtDNA variation. This has often lead to the analysis of convenience samples collected for other purposes, which might not meet the quality requirement of random sampling for mtDNA data sets. Here, we introduce an additional quality control means that deals with one aspect of this limitation: by combining autosomal short tandem repeat (STR) marker with mtDNA information, it helps to avoid the bias introduced by related individuals included in the same (small) sample. By STR analysis of individuals sharing their mitochondrial haplotype, pedigree construction and subsequent software-assisted calculation of likelihood ratios based on the allele frequencies found in the population, closely maternally related individuals can be identified and excluded. We also discuss scenarios that allow related individuals in the same set. An ideal population sample would be representative for its population: this new approach represents another contribution towards this goal. PMID:21067986

  2. Satisfaction survey with DNA cards method to collect genetic samples for pharmacogenetics studies

    PubMed Central

    Vidal-Taboada, Jose M; Cucala, Mercedes; Mas Herrero, Sergio; Lafuente, Amalia; Cobos, Albert

    2006-01-01

    Background Pharmacogenetic studies are essential in understanding the interindividual variability of drug responses. DNA sample collection for genotyping is a critical step in genetic studies. A method using dried blood samples from finger-puncture, collected on DNA-cards, has been described as an alternative to the usual venepuncture technique. The purpose of this study is to evaluate the implementation of the DNA cards method in a multicentre clinical trial, and to assess the degree of investigators' satisfaction and the acceptance of the patients perceived by the investigators. Methods Blood samples were collected on DNA-cards. The quality and quantity of DNA recovered were analyzed. Investigators were questioned regarding their general interest, previous experience, safety issues, preferences and perceived patient satisfaction. Results 151 patients' blood samples were collected. Genotyping of GST polymorphisms was achieved in all samples (100%). 28 investigators completed the survey. Investigators perceived patient satisfaction as very good (60.7%) or good (39.3%), without reluctance to finger puncture. Investigators preferred this method, which was considered safer and better than the usual methods. All investigators would recommend using it in future genetic studies. Conclusion Within the clinical trial setting, the DNA-cards method was very well accepted by investigators and patients (in perception of investigators), and was preferred to conventional methods due to its ease of use and safety. PMID:16681846

  3. Using DNA-Stable Isotope Probing to Identify MTBE- and TBA-Degrading Microorganisms in Contaminated Groundwater

    PubMed Central

    Key, Katherine C.; Sublette, Kerry L.; Duncan, Kathleen; Mackay, Douglas M.; Scow, Kate M.; Ogles, Dora

    2014-01-01

    Although the anaerobic biodegradation of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA) has been documented in the laboratory and the field, knowledge of the microorganisms and mechanisms involved is still lacking. In this study, DNA-stable isotope probing (SIP) was used to identify microorganisms involved in anaerobic fuel oxygenate biodegradation in a sulfate-reducing MTBE and TBA plume. Microorganisms were collected in the field using Bio-Sep® beads amended with 13C5-MTBE, 13C1-MTBE (only methoxy carbon labeled), or13C4-TBA. 13C-DNA and 12C-DNA extracted from the Bio-Sep beads were cloned and 16S rRNA gene sequences were used to identify the indigenous microorganisms involved in degrading the methoxy group of MTBE and the tert-butyl group of MTBE and TBA. Results indicated that microorganisms were actively degrading 13C-labeled MTBE and TBA in situ and the 13C was incorporated into their DNA. Several sequences related to known MTBE- and TBA-degraders in the Burkholderiales and the Sphingomonadales orders were detected in all three13C clone libraries and were likely to be primary degraders at the site. Sequences related to sulfate-reducing bacteria and iron-reducers, such as Geobacter and Geothrix, were only detected in the clone libraries where MTBE and TBA were fully labeled with 13C, suggesting that they were involved in processing carbon from the tert-butyl group. Sequences similar to the Pseudomonas genus predominated in the clone library where only the methoxy carbon of MTBE was labeled with 13C. It is likely that members of this genus were secondary degraders cross-feeding on 13C-labeled metabolites such as acetate. PMID:25525320

  4. Comparison of three different DNA extraction methods from a highly degraded biological material.

    PubMed

    Kuś, M; Ossowski, A; Zielińska, G

    2016-05-01

    The identification of unknown victims is one of the most challenging tasks faced by forensic medicine. This is due to the rapid decomposition of tissues, beginning at the moment of death and caused by released enzymes and microbial activity. Decay is directly associated with the decomposition of soft tissues and also the degradation of genetic material inside cells. Decomposition rates vary depending on a number of environmental factors, including temperature, humidity, season, and soil properties. Decomposition also differs between bodies left in the open air or buried. To date, forensic medicine has identified mainly people who were the victims of various types of criminal offences. However, with advances in identification methods, increasingly frequent attempts are made to identify the victims of armed conflicts, crimes of totalitarian regimes, or genocide. The aim of the study was to compare three different methods for the extraction of nuclear DNA from material considered in forensic medicine as difficult to handle, i.e. fragments of bones and teeth, and to determine the performance of these methods and their suitability for identification procedures.

  5. The phylogeny and evolution of deoxyribonuclease II: An enzyme essential for lysosomal DNA degradation

    PubMed Central

    Shpak, Max; Kugelman, Jeffrey R.; Varela-Ramirez, Armando; Aguilera, Renato J.

    2008-01-01

    Deoxyribonuclease II (DNase II) is an endonuclease with optimal activity at low pH, localized within the lysosomes of higher eukaryotes. The origin of this enzyme remains in dispute, and its phylogenetic distribution leaves many questions about its subsequent evolutionary history open. Earlier studies have documented its presence in various metazoans, as well as in Dictyostelium, Trichomonas and, anomalously, a single genus of bacteria (Burkholderia). This study makes use of searches of the genomes of various organisms against known DNase II query sequences, in order to determine the likely point of origin of this enzyme among cellular life forms. Its complete absence from any other bacteria makes prokaryotic origin unlikely. Convincing evidence exists for DNase II homologs in Alveolates such as Paramecium, Heterokonts such as diatoms and water molds, and even tentative matches in green algae. Apparent absences include red algae, plants, fungi, and a number of parasitic organisms. Based on this phylogenetic distribution and hypotheses of eukaryotic relationships, the most probable explanation is that DNase II has been subject to multiple losses. The point of origin is debatable, though its presence in Trichomonas and perhaps in other evolutionarily basal “Excavate” protists such as Reclinomonas, strongly support the hypothesis that DNase II arose as a plesiomorphic trait in eukaryotes. It probably evolved together with phagocytosis, specifically to facilitate DNA degradation and bacteriotrophy. The various absences in many eukaryotic lineages are accounted for by loss of phagotrophic function in intracellular parasites, in obligate autotrophs, and in saprophytes. PMID:18226927

  6. Non-degradative Intracellular Trafficking of Highly Compacted Polymeric DNA Nanoparticles

    PubMed Central

    Kim, Anthony J.; Boylan, Nicholas J.; Suk, Jung Soo; Lai, Samuel K.; Hanes, Justin

    2011-01-01

    Highly compacted DNA nanoparticles (DNPs) composed of polyethylene glycol linked to a 30-mer of poly-L-lysine via a single cysteine residue (CK30PEG) have previously been shown to provide efficient gene delivery to the brain, eyes and lungs. In this study, we used a combination of flow cytometry, high-resolution live-cell confocal microscopy, and multiple particle tracking (MPT) to investigate the intracellular trafficking of highly compacted CK30PEG DNPs made using two different molecular weights of PEG, CK30PEG10k and CK30PEG5k. We found that PEG MW did not have a major effect on particle morphology nor nanoparticle intracellular transport. CK30PEG10k and CK30PEG5k DNPs both entered human bronchial epithelial (BEAS-2B) cells via a caveolae-mediated pathway, bypassing degradative endolysosomal trafficking. Both nanoparticle formulations were found to rapidly accumulate in the perinuclear region of cells within 2 h, 37 ± 19 % and 47 ± 8 % for CK30PEG10k and CK30PEG5k, respectively. CK30PEG10k and CK30PEG5k DNPs moved within live cells at average velocities of 0.09 ± 0.04 µm/s and 0.11 ± 0.04 µm/s, respectively, in good agreement with reported values for caveolae. These findings show that highly compacted DNPs employ highly regulated trafficking mechanisms similar to biological pathogens to target specific intracellular compartments. PMID:22079809

  7. Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA Samples

    PubMed Central

    Rhee, Minsoung; Light, Yooli K.; Meagher, Robert J.; Singh, Anup K.

    2016-01-01

    Multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology. PMID:27144304

  8. Digital droplet multiple displacement amplification (ddMDA) for whole genome sequencing of limited DNA samples

    SciTech Connect

    Rhee, Minsoung; Light, Yooli K.; Meagher, Robert J.; Singh, Anup K.; Kumar-Sinha, Chandan

    2016-05-04

    Here, multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.

  9. Digital droplet multiple displacement amplification (ddMDA) for whole genome sequencing of limited DNA samples

    DOE PAGES

    Rhee, Minsoung; Light, Yooli K.; Meagher, Robert J.; ...

    2016-05-04

    Here, multiple displacement amplification (MDA) is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples) before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA) technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently,more » the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet), ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli) compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.« less

  10. Y-STRs in forensic medicine: DNA analysis in semen samples of azoospermic individuals.

    PubMed

    Soares-Vieira, José Arnaldo; Billerbeck, Ana Elisa Correia; Iwamura, Edna Sadayo Miazato; Zampieri, Ricardo Andrade; Gattás, Gilka Jorge Fígaro; Munoz, Daniel Romero; Hallak, Jorge; Mendonca, Berenice Bilharinho; Lucon, Antonio Marmo

    2007-05-01

    The incidence of rape has increased, especially in metropolitan areas, such as the city of São Paulo. In Brazil, studies about it have shown that the majority of this type of crime is committed by the relatives and persons close to the victim. This has made the crime more difficult to be denounced, as only 10% of the cases are reported to competent police authorities. Usually, cytological exams are carried out in sex crime investigations. The difficulty in showing the presence of spermatozoa is frequent, but it does not exclude the presence of male DNA. The absence of spermatozoa in material collected from rape victims can be due to several factors, including the fact that the agressor suffers from azoospermia. This condition can be the result of a successful vasectomy. As the majority of DNA in the ejaculation sample is from spermatozoa, there is much less DNA to be analyzed. This study presents the application of Y-STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393) in DNA analysis of sperm samples from 105 vasectomized men. The study demonstrated a great variation in DNA concentration. DNA extraction and amplification was possible in all sperm samples even in the absence of spermatozoa. The same profile was observed, for each individual, from DNA extracted from blood, pre- and postvasectomy semen samples. The use of markers specific for Y chromosome in sex crime cases, especially in the absence of spermatozoa, is very important, mainly because in most situations there is a small quantity of the agressor's DNA in the medium and a large quantity of the victim's DNA.

  11. Post-coital vaginal sampling with nylon flocked swabs improves DNA typing.

    PubMed

    Benschop, Corina C G; Wiebosch, Danielle C; Kloosterman, Ate D; Sijen, Titia

    2010-02-01

    In the examination of sexual assault cases, DNA typing of vaginal samples mostly occurs after differential DNA extraction. Notwithstanding the differential extraction method, the DNA profiles from the seminal fraction often show the male alleles at low-level in combination with female alleles. This unfavorable ratio male to female DNA is due to a limited amount of sperm cells and an overwhelming quantity of female cells. In this study, we compared standard cotton and nylon flocked swabs for post-coital vaginal sampling. Twelve couples donated 88 vaginal swabs - 44 cotton, 44 nylon flocked - which were taken with a time since intercourse (TSI) up to 84 h. These vaginal swabs were sorted into categories on the basis of the TSI and submitted to (1) microscopic examination for the presence of male cells, (2) presumptive tests for the detection of seminal fluid and (3) DNA typing. Cellular elution was found to be 6-fold more efficient from the nylon flocked swabs. This makes microscopic analysis less time consuming as the higher cell yield and better cell morphology simplify detection of male cells. Both swab types reveal similar results regarding presumptive tests and male DNA typing. Positive presumptive tests (RSID-semen and PSA) were obtained up to 60 h TSI and male autosomal profiles up to 72 h TSI. Interestingly, over 50% of the samples negative for both presumptive tests resulted in informative male STR profiles. After differential extraction, less DNA was left on the nylon flocked swabs and more male DNA was isolated. Our results imply that the use of nylon flocked swabs for vaginal sampling will improve microscopic analysis and DNA typing in the medical forensic investigation of sexual assault cases.

  12. Significant finding of Bordetella holmesii DNA in nasopharyngeal samples from French patients with suspected pertussis.

    PubMed

    Njamkepo, Elisabeth; Bonacorsi, Stéphane; Debruyne, Monique; Gibaud, Sophie Anne; Guillot, Sophie; Guiso, Nicole

    2011-12-01

    Pertussis is routinely diagnosed with real-time PCR based on insertion sequence IS481, which is not specific for Bordetella pertussis. We conducted a retrospective study using real-time PCRs specific for Bordetella pertussis and for Bordetella holmesii on 177 samples positive for IS481 PCR. Bordetella holmesii DNA was detected in 20.3% samples collected from adolescents and adults.

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

    PubMed

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

    2009-02-03

    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.

  14. STR DNA typing: increased sensitivity and efficient sample consumption using reduced PCR reaction volumes.

    PubMed

    Leclair, Benoît; Sgueglia, Joanne B; Wojtowicz, Patricia C; Juston, Ann C; Frégeau, Chantal J; Fourney, Ron M

    2003-09-01

    Improvements in detection limits/sensitivity and lower sample consumption are potential benefits of reducing PCR reaction volumes used in forensic DNA typing of crime scene samples. This premise was studied first with experimental mixtures and a nine-loci megaplex, which demonstrated stochiometric amplification and accurate detection. Next, adjudicated casework samples were subjected to amplification under 15 different template DNA to PCR reaction volume ratios. Reduction of PCR reaction volume and DNA down to 10 microL and 0.500 ng, respectively, produced identical profiles with the same signal intensity and heterozygous allele peak height ratio (HR). Reduction to 5 microL and 0.063 ng yielded HR values that were slightly affected in one to three STR loci. PCR reaction volume reduction can enhance detection and sensitivity while reducing the consumption of irreplaceable crime scene samples.

  15. Degradation and half-life of DNA present in biomass from a genetically-modified organism during land application.

    PubMed

    Halter, Mathew C; Zahn, James A

    2017-02-01

    White biotechnology has made a positive impact on the chemical industry by providing safer, more efficient chemical manufacturing processes that have reduced the use of toxic chemicals, harsh reaction conditions, and expensive metal catalysts, which has improved alignment with the principles of Green Chemistry. The genetically-modified (GM) biocatalysts that are utilized in these processes are typically separated from high-value products and then recycled, or eliminated. Elimination routes include disposal in sanitary landfills, incineration, use as a fuel, animal feed, or reuse as an agricultural soil amendment or other value-added products. Elimination routes that have the potential to impact the food chain or environment have been more heavily scrutinized for the fate and persistence of biological products. In this study, we developed and optimized a method for monitoring the degradation of strain-specific DNA markers from a genetically-modified organism (GMO) used for the commercial production of 1,3-propanediol. Laboratory and field tests showed that a marker for heterologous DNA in the GM organism was no longer detectable by end-point polymerase chain reaction (PCR) after 14 days. The half-life of heterologous DNA was increased by 17% (from 42.4 to 49.7 h) after sterilization of the soil from a field plot, which indicated that abiotic factors were important in degradation of DNA under field conditions. There was no evidence for horizontal transfer of DNA target sequences from the GMO to viable organisms present in the soil.

  16. Modification of extraction method for community DNA isolation from salt affected compact wasteland soil samples.

    PubMed

    Zaveri, Purvi; Patel, Rushika; Patel, Meghavi; Sarodia, Devki; Munshi, Nasreen S

    2017-01-01

    To overcome the issue of interferences by salt and compactness in release of bacterial cell required for lysis, method described by Yeates et al. (1998), was optimized for isolation of genomic material (Deoxyribo Nucleic Acid, DNA) from soil microbial community by addition of Al(NH4)SO4. Very low total viable count was observed in the samples tested and hence use of higher amount of soil is required primarily for DNA isolation from wasteland soils. The method proves itself efficient where commercially available bead beating and enzymatic lysis methods could not give isolation of any amount of community genomic DNA due to compact nature and salt concentrations present in soil. •The protocol was found efficient for soil samples with high clay content for microbial community DNA extraction.•Variation in lysis incubation and amount of soil may help with soil samples containing low microbial population.•Addition of Al(NH4)SO4 is crucial step in humic acid removal from extracted DNA samples for soil samples containing high salinity and clay particles.

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

    PubMed Central

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

    2013-01-01

    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. PMID:23409092

  18. Nondestructive sampling of human skeletal remains yields ancient nuclear and mitochondrial DNA.

    PubMed

    Bolnick, Deborah A; Bonine, Holly M; Mata-Míguez, Jaime; Kemp, Brian M; Snow, Meradeth H; LeBlanc, Steven A

    2012-02-01

    Museum curators and living communities are sometimes reluctant to permit ancient DNA (aDNA) studies of human skeletal remains because the extraction of aDNA usually requires the destruction of at least some skeletal material. Whether these views stem from a desire to conserve precious materials or an objection to destroying ancestral remains, they limit the potential of aDNA research. To help address concerns about destructive analysis and to minimize damage to valuable specimens, we describe a nondestructive method for extracting DNA from ancient human remains. This method can be used with both teeth and bone, but it preserves the structural integrity of teeth much more effectively than that of bone. Using this method, we demonstrate that it is possible to extract both mitochondrial and nuclear DNA from human remains dating between 300 BC and 1600 AD. Importantly, the method does not expose the remains to hazardous chemicals, allowing them to be safely returned to curators, custodians, and/or owners of the samples. We successfully amplified mitochondrial DNA from 90% of the individuals tested, and we were able to analyze 1-9 nuclear loci in 70% of individuals. We also show that repeated nondestructive extractions from the same tooth can yield amplifiable mitochondrial and nuclear DNA. The high success rate of this method and its ability to yield DNA from samples spanning a wide geographic and temporal range without destroying the structural integrity of the sampled material may make possible the genetic study of skeletal collections that are not available for destructive analysis.

  19. Study of DNA damage with a new system for irradiation of samples in a nuclear reactor.

    PubMed

    Gual, Maritza R; Milian, Felix M; Deppman, Airton; Coelho, Paulo R P

    2011-02-01

    In this paper, we report results of a quantitative analysis of the effects of neutrons on DNA, and, specifically, the production of simple and double breaks of plasmid DNA in aqueous solutions with different concentrations of free-radical scavengers. The radiation damage to DNA was evaluated by electrophoresis through agarose gels. The neutron and gamma doses were measured separately with thermoluminescent detectors. In this work, we have also demonstrated usefulness of a new system for positioning and removing samples in channel BH#3 of the IEA-R1 reactor at the Instituto de Pesquisas Energéticas e Nucleares (Brazil) without necessity of interrupting the reactor operation.

  20. Environmental DNA from Seawater Samples Correlate with Trawl Catches of Subarctic, Deepwater Fishes

    PubMed Central

    Thomsen, Philip Francis; Møller, Peter Rask; Sigsgaard, Eva Egelyng; Knudsen, Steen Wilhelm; Jørgensen, Ole Ankjær; Willerslev, Eske

    2016-01-01

    Remote polar and deepwater fish faunas are under pressure from ongoing climate change and increasing fishing effort. However, these fish communities are difficult to monitor for logistic and financial reasons. Currently, monitoring of marine fishes largely relies on invasive techniques such as bottom trawling, and on official reporting of global catches, which can be unreliable. Thus, there is need for alternative and non-invasive techniques for qualitative and quantitative oceanic fish surveys. Here we report environmental DNA (eDNA) metabarcoding of seawater samples from continental slope depths in Southwest Greenland. We collected seawater samples at depths of 188–918 m and compared seawater eDNA to catch data from trawling. We used Illumina sequencing of PCR products to demonstrate that eDNA reads show equivalence to fishing catch data obtained from trawling. Twenty-six families were found with both trawling and eDNA, while three families were found only with eDNA and two families were found only with trawling. Key commercial fish species for Greenland were the most abundant species in both eDNA reads and biomass catch, and interpolation of eDNA abundances between sampling sites showed good correspondence with catch sizes. Environmental DNA sequence reads from the fish assemblages correlated with biomass and abundance data obtained from trawling. Interestingly, the Greenland shark (Somniosus microcephalus) showed high abundance of eDNA reads despite only a single specimen being caught, demonstrating the relevance of the eDNA approach for large species that can probably avoid bottom trawls in most cases. Quantitative detection of marine fish using eDNA remains to be tested further to ascertain whether this technique is able to yield credible results for routine application in fisheries. Nevertheless, our study demonstrates that eDNA reads can be used as a qualitative and quantitative proxy for marine fish assemblages in deepwater oceanic habitats. This relates

  1. Environmental DNA from Seawater Samples Correlate with Trawl Catches of Subarctic, Deepwater Fishes.

    PubMed

    Thomsen, Philip Francis; Møller, Peter Rask; Sigsgaard, Eva Egelyng; Knudsen, Steen Wilhelm; Jørgensen, Ole Ankjær; Willerslev, Eske

    2016-01-01

    Remote polar and deepwater fish faunas are under pressure from ongoing climate change and increasing fishing effort. However, these fish communities are difficult to monitor for logistic and financial reasons. Currently, monitoring of marine fishes largely relies on invasive techniques such as bottom trawling, and on official reporting of global catches, which can be unreliable. Thus, there is need for alternative and non-invasive techniques for qualitative and quantitative oceanic fish surveys. Here we report environmental DNA (eDNA) metabarcoding of seawater samples from continental slope depths in Southwest Greenland. We collected seawater samples at depths of 188-918 m and compared seawater eDNA to catch data from trawling. We used Illumina sequencing of PCR products to demonstrate that eDNA reads show equivalence to fishing catch data obtained from trawling. Twenty-six families were found with both trawling and eDNA, while three families were found only with eDNA and two families were found only with trawling. Key commercial fish species for Greenland were the most abundant species in both eDNA reads and biomass catch, and interpolation of eDNA abundances between sampling sites showed good correspondence with catch sizes. Environmental DNA sequence reads from the fish assemblages correlated with biomass and abundance data obtained from trawling. Interestingly, the Greenland shark (Somniosus microcephalus) showed high abundance of eDNA reads despite only a single specimen being caught, demonstrating the relevance of the eDNA approach for large species that can probably avoid bottom trawls in most cases. Quantitative detection of marine fish using eDNA remains to be tested further to ascertain whether this technique is able to yield credible results for routine application in fisheries. Nevertheless, our study demonstrates that eDNA reads can be used as a qualitative and quantitative proxy for marine fish assemblages in deepwater oceanic habitats. This relates

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

    PubMed

    Morin, P A; Chambers, K E; Boesch, C; Vigilant, L

    2001-07-01

    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 DNA in low DNA concentration extracts from noninvasive samples. We describe the range of DNA amounts obtained from chimpanzee faeces and shed hair samples and formulate a new efficient approach for accurate microsatellite genotyping. Prescreening of extracts for DNA quantity is recommended for sorting of samples for likely success and reliability. Repetition of results remains extensive for analysis of microsatellite amplifications beginning from low starting amounts of DNA, but is reduced for those with higher DNA content.

  3. Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA

    PubMed Central

    Minczuk, Michal; Papworth, Monika A.; Miller, Jeffrey C.; Murphy, Michael P.; Klug, Aaron

    2008-01-01

    The selective degradation of mutated mitochondrial DNA (mtDNA) molecules is a potential strategy to re-populate cells with wild-type (wt) mtDNA molecules and thereby alleviate the defective mitochondrial function that underlies mtDNA diseases. Zinc finger nucleases (ZFNs), which are nucleases conjugated to a zinc-finger peptide (ZFP) engineered to bind a specific DNA sequence, could be useful for the selective degradation of particular mtDNA sequences. Typically, pairs of complementary ZFNs are used that heterodimerize on the target DNA sequence; however, conventional ZFNs were ineffective in our system. To overcome this, we created single-chain ZFNs by conjugating two FokI nuclease domains, connected by a flexible linker, to a ZFP with an N-terminal mitochondrial targeting sequence. Here we show that these ZFNs are efficiently transported into mitochondria in cells and bind mtDNA in a sequence-specific manner discriminating between two 12-bp long sequences that differ by a single base pair. Due to their selective binding they cleave dsDNA at predicted sites adjacent to the mutation. When expressed in heteroplasmic cells containing a mixture of mutated and wt mtDNA these ZFNs selectively degrade mutated mtDNA, thereby increasing the proportion of wt mtDNA molecules in the cell. Therefore, mitochondria-targeted single-chain ZFNs are a promising candidate approach for the treatment of mtDNA diseases. PMID:18511461

  4. PE-Swab Direct STR Amplification of Forensic Touch DNA Samples.

    PubMed

    Liu, Jason Y

    2015-05-01

    The PE-Swab direct STR amplification workflow was developed to process low-level "touch DNA" samples. In this workflow, a forensic sample is first collected on a 4-mm PE-Swab (a novel sample collection device); two 2-mm punches containing collected samples are then generated from the PE-Swab and directly amplified for STR typing. Compared to the conventional STR workflow, which involves DNA extraction, purification, and elution volume reduction, the PE-Swab direct STR amplification workflow does not require sample preparation and takes <60 sec before a touch sample is ready for STR amplification. Because there is no DNA loss due to sample preparation, the PE-Swab workflow is more sensitive than the conventional STR workflow. The average peak height per sample obtained by the PE-swab workflow is 3 times higher than that from the conventional workflow with both low-level single source and two-contributor mixture samples tested in this study.

  5. Ancient mitochondrial DNA from Malaysian hair samples: some indications of Southeast Asian population movements.

    PubMed

    Ricaut, François-X; Bellatti, M; Lahr, Marta Mirazon

    2006-01-01

    The late Pleistocene and early Holocene population history of Southeast Asia is not well-known. Our study provides new data on mitochondrial DNA (mtDNA) lineages of the aboriginal inhabitants of the Malay Peninsula, and through an extensive comparison to the known mtDNA diversity in Southeast and East Asia, provides some new insights into the origins and historical geography of certain mtDNA lineages in the region. We extracted DNA from hair samples (dating back 100 years) preserved in the Duckworth Collection and belonging to two Peninsular Malaysian individuals identified as "Negrito." Ancient DNA was analyzed by sequencing hypervariable region I (HVS-I) of the mtDNA control region and the mtDNA region V length polymorphism. The results show that the maternal lineages of these individuals belong to a recently defined haplogroup B sub-branch called B4c2. A comparison of mitochondrial haplotypes and haplogroups with those of 10,349 East Asian individuals indicates their very restricted geographical distribution (southwestern China, Southeast Asia Peninsula, and Indonesia). Recalculation of the B4c2 age across all of East Asia ( approximately 13,000 years) and in different subregions/populations suggests its rapid diffusion in Southeast Asia between the end of the Last Glacial Maximum and the Neolithic expansion of the Holocene.

  6. A high-throughput bioimaging study to assess the impact of chitosan-based nanoparticle degradation on DNA delivery performance.

    PubMed

    Gomes, Carla P; Varela-Moreira, Aida; Leiro, Victoria; Lopes, Cátia D F; Moreno, Pedro M D; Gomez-Lazaro, Maria; Pêgo, Ana P

    2016-12-01

    By using imaging flow cytometry as a powerful statistical high-throughput technique we investigated the impact of degradation on the biological performance of trimethyl chitosan (TMC)-based nanoparticles (NPs). In order to achieve high transfection efficiencies, a precise balance between NP stability and degradation must occur. We altered the biodegradation rate of the TMC NPs by varying the degree of acetylation (DA) of the polymer (DA ranged from 4 to 21%), giving rise to NPs with different enzymatic degradation profiles. While this parameter did not affect NP size, charge or ability to protect plasmid DNA, NPs based on TMC with an intermediate DA (16%) showed the highest transfection efficiency. Subsequently, by means of a single quantitative technique, we were able to follow, for each tested formulation, major steps of the NP-mediated gene delivery process - NP cell membrane association, internalization and intracellular trafficking, including plasmid DNA transport towards the nucleus. NP cytotoxicity was also possible to determine by quantification of cell apoptosis. Overall, the obtained data revealed that the biodegradation rate of these NPs affects their intracellular trafficking and, consequently, their efficiency to transfect cells. Thus, one can use the polymer DA to modulate the NPs towards attaining different degradation rates and tune their bioactivity according to the desired application. Furthermore, this novel technical approach revealed to be a valuable tool for the initial steps of nucleic acid vector design.

  7. Study of microtip-based extraction and purification of DNA from human samples for portable devices

    NASA Astrophysics Data System (ADS)

    Fotouhi, Gareth

    DNA sample preparation is essential for genetic analysis. However, rapid and easy-to-use methods are a major challenge to obtaining genetic information. Furthermore, DNA sample preparation technology must follow the growing need for point-of-care (POC) diagnostics. The current use of centrifuges, large robots, and laboratory-intensive protocols has to be minimized to meet the global challenge of limited access healthcare by bringing the lab to patients through POC devices. To address these challenges, a novel extraction method of genomic DNA from human samples is presented by using heat-cured polyethyleneimine-coated microtips generating a high electric field. The microtip extraction method is based on recent work using an electric field and capillary action integrated into an automated device. The main challenges to the method are: (1) to obtain a stable microtip surface for the controlled capture and release of DNA and (2) to improve the recovery of DNA from samples with a high concentration of inhibitors, such as human samples. The present study addresses these challenges by investigating the heat curing of polyethyleneimine (PEI) coated on the surface of the microtip. Heat-cured PEI-coated microtips are shown to control the capture and release of DNA. Protocols are developed for the extraction and purification of DNA from human samples. Heat-cured PEI-coated microtip methods of DNA sample preparation are used to extract genomic DNA from human samples. It is discovered through experiment that heat curing of a PEI layer on a gold-coated surface below 150°C could inhibit the signal of polymerase chain reaction (PCR). Below 150°C, the PEI layer is not completely cured and dissolved off the gold-coated surface. Dissolved PEI binds with DNA to inhibit PCR. Heat curing of a PEI layer above 150°C on a gold-coated surface prevents inhibition to PCR and gel electrophoresis. In comparison to gold-coated microtips, the 225°C-cured PEI-coated microtips improve the

  8. Applicability of the ParaDNA(®) Screening System to Seminal Samples.

    PubMed

    Tribble, Nicholas D; Miller, Jamie A D; Dawnay, Nick; Duxbury, Nicola J

    2015-05-01

    Seminal fluid represents a common biological material recovered from sexual assault crime scenes. Such samples can be prescreened using different techniques to determine cell type and relative amount before submitting for full STR profiling. The ParaDNA(®) Screening System is a novel forensic test which identifies the presence of DNA through amplification and detection of two common STR loci (D16S539 and TH01) and the Amelogenin marker. The detection of the Y allele in samples could provide a useful tool in the triage and submission of sexual assault samples by enforcement authorities. Male template material was detected on a range of common sexual assault evidence items including cotton pillow cases, condoms, swab heads and glass surfaces and shows a detection limit of 1 in 1000 dilution of neat semen. These data indicate this technology has the potential to be a useful tool for the detection of male donor DNA in sexual assault casework.

  9. Quantitative Field Testing Heterodera glycines from Metagenomic DNA Samples Isolated Directly from Soil under Agronomic Production

    PubMed Central

    Li, Yan; Lawrence, Gary W.; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P.

    2014-01-01

    A quantitative PCR procedure targeting the Heterodera glycines ortholog of the Caenorhabditis elegans uncoordinated-78 gene was developed. The procedure estimated the quantity of H. glycines from metagenomic DNA samples isolated directly from field soil under agronomic production. The estimation of H. glycines quantity was determined in soil samples having other soil dwelling plant parasitic nematodes including Hoplolaimus, predatory nematodes including Mononchus, free-living nematodes and biomass. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from field soil. PMID:24587100

  10. Chromatin-associated degradation is defined by UBXN-3/FAF1 to safeguard DNA replication fork progression

    PubMed Central

    Franz, André; Pirson, Paul A.; Pilger, Domenic; Halder, Swagata; Achuthankutty, Divya; Kashkar, Hamid; Ramadan, Kristijan; Hoppe, Thorsten

    2016-01-01

    The coordinated activity of DNA replication factors is a highly dynamic process that involves ubiquitin-dependent regulation. In this context, the ubiquitin-directed ATPase CDC-48/p97 recently emerged as a key regulator of chromatin-associated degradation in several of the DNA metabolic pathways that assure genome integrity. However, the spatiotemporal control of distinct CDC-48/p97 substrates in the chromatin environment remained unclear. Here, we report that progression of the DNA replication fork is coordinated by UBXN-3/FAF1. UBXN-3/FAF1 binds to the licensing factor CDT-1 and additional ubiquitylated proteins, thus promoting CDC-48/p97-dependent turnover and disassembly of DNA replication factor complexes. Consequently, inactivation of UBXN-3/FAF1 stabilizes CDT-1 and CDC-45/GINS on chromatin, causing severe defects in replication fork dynamics accompanied by pronounced replication stress and eventually resulting in genome instability. Our work identifies a critical substrate selection module of CDC-48/p97 required for chromatin-associated protein degradation in both Caenorhabditis elegans and humans, which is relevant to oncogenesis and aging. PMID:26842564

  11. A sample preparation protocol for quantification of radiolabeled nucleoside incorporation into DNA by accelerator mass spectrometry

    NASA Astrophysics Data System (ADS)

    Hah, Sang Soo; Mundt, Janna M.; Ubick, Esther A.; Turteltaub, Kenneth W.; Gregg, Jeff P.; Henderson, Paul T.

    2007-06-01

    A general protocol is described for measuring the incorporation of radiocarbon-labeled 2‧-deoxynucleosides into DNA using accelerator mass spectrometry (AMS). This technology provides attomole (10-18 mol) sensitivity, with detection limits for DNA analysis in the range of one 14C atom per 1011-1012 total carbons. In practice this corresponds to approximately 1 labeled nucleoside per 1011 normal bases. A key aspect of the method is the use of precautions aimed at prevention of artifactual DNA oxidation during the sample preparation by the use of antioxidants and chaotropic salts during the DNA isolation. In principle, any type of appropriately labeled nucleoside derivative can be studied using the described protocol, provided that there is incorporation of the deoxynucleoside into DNA. We demonstrated this protocol using MCF-7 human breast cancer cells and a mouse model for mammary carcinoma, which we dosed with 14C-labeled 2‧-deoxyguanosine (dG) and 14C-labeled 7,8-dihydro-8-oxo-2‧-deoxyguanosine (8-oxodG). The nucleoside 8-oxodG is a ubiquitous compound that forms in cells by the reaction of dG with reactive oxygen species which has been associated with numerous disease, carcinogenesis and aging. DNA from cells treated with 14C-labeled nucleosides was isolated and prepared for analysis by AMS in order to measure the DNA-bound radioactivity. The method allows the generation of reliable and sufficient yields of pure DNA from human cells and animal tissues for analysis of radiocarbon levels. Ultimately, this protocol will be applied to understanding the role of modified nucleoside incorporation into DNA in cancer initiation and progression, but could also be used to study any DNA metabolism process where 14C-labeled nucleosides are used.

  12. 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

    SciTech Connect

    Eckels, D.E.; Hass, W.J.

    1989-05-30

    A sample transport, sample introduction, and flame excitation system is described for spectrometric analysis of high temperature gas streams which eliminates degradation of the sample stream by condensation losses. 4 figs.

  13. Sensitive PCR analysis of animal tissue samples for fragments of endogenous and transgenic plant DNA.

    PubMed

    Nemeth, Anne; Wurz, Andreas; Artim, Lori; Charlton, Stacy; Dana, Greg; Glenn, Kevin; Hunst, Penny; Jennings, James; Shilito, Ray; Song, Ping

    2004-10-06

    An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples.

  14. Sequence analysis of the canine mitochondrial DNA control region from shed hair samples in criminal investigations.

    PubMed

    Berger, C; Berger, B; Parson, W

    2012-01-01

    In recent years, evidence from domestic dogs has increasingly been analyzed by forensic DNA testing. Especially, canine hairs have proved most suitable and practical due to the high rate of hair transfer occurring between dogs and humans. Starting with the description of a contamination-free sample handling procedure, we give a detailed workflow for sequencing hypervariable segments (HVS) of the mtDNA control region from canine evidence. After the hair material is lysed and the DNA extracted by Phenol/Chloroform, the amplification and sequencing strategy comprises the HVS I and II of the canine control region and is optimized for DNA of medium-to-low quality and quantity. The sequencing procedure is based on the Sanger Big-dye deoxy-terminator method and the separation of the sequencing reaction products is performed on a conventional multicolor fluorescence detection capillary electrophoresis platform. Finally, software-aided base calling and sequence interpretation are addressed exemplarily.

  15. [Infrared Spectrum Studies of Hydrocarbon Generation and Structure Evolution of Peat Samples During Pyrolysis and Microbial Degradation].

    PubMed

    Bao, Yuan; Ju, Yi-wen; Wei, Chong-tao; Wang, Chao-yong; Li, Xiao-shi

    2015-03-01

    Hydrocarbon generation and structural evolution would be occurred in the process of from coal-forming material (i. e. peat sample) transforming to the coal. While Fourier Transform Infrared Spectroscopy (FTIR) have a special advantages in analyzing molecular structure of samples. For understanding the characteristics of hydrocarbon generation and structural evolution of coal-forming material during the process of pyrolysis and microbial degradation, based on the physical simulation experiments of closed pyrolysis and anaerobic microbial degradation, the generation potential of thermogenic gas and biogenic gas were studied in this paper, and characteristics of molecular structure evolution and its mechanism was analyzed by FTIR technology. Results show that cumulative gas yields of hydrocarbon gases (mainly for methane) increased with experiment temperature. The gas yield of non-hydrocarbon gas (mainly for CO2) exhibited two peaks at 250 and 375 degrees C. The degradation ability of anaerobe on coal samples weakened with the maturity increasing and there was no gas generation on the pyrolysis samples with maturity from 1.6% to 1.8%. After pyrolysis, the content of hydroxyl in peat sample decreased first and then increased with the pyrolysis temperature increasing. The content of aldehyde carbonyl, methylene and phosphate reduced. The content of aromatic esters decreased with nonlinear. The bone of S-O in stretching vibration appeared after 350 degrees C and its content increased with temperature. This shows that the sulfocompound restrains the activity of methanogenic bacteria. After degradation by anaerobe, the relative content of hydroxyl, aldehyde carbonyl, aromatic esters, methylene and phosphate in peat sample dropped significantly. It is shown that the intermolecular force between these groups weakened.

  16. Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition

    PubMed Central

    Munk, Patrick; Lukjancenko, Oksana; Priemé, Anders; Aarestrup, Frank M.

    2016-01-01

    ABSTRACT Explorations of complex microbiomes using genomics greatly enhance our understanding about their diversity, biogeography, and function. The isolation of DNA from microbiome specimens is a key prerequisite for such examinations, but challenges remain in obtaining sufficient DNA quantities required for certain sequencing approaches, achieving accurate genomic inference of microbiome composition, and facilitating comparability of findings across specimen types and sequencing projects. These aspects are particularly relevant for the genomics-based global surveillance of infectious agents and antimicrobial resistance from different reservoirs. Here, we compare in a stepwise approach a total of eight commercially available DNA extraction kits and 16 procedures based on these for three specimen types (human feces, pig feces, and hospital sewage). We assess DNA extraction using spike-in controls and different types of beads for bead beating, facilitating cell lysis. We evaluate DNA concentration, purity, and stability and microbial community composition using 16S rRNA gene sequencing and for selected samples using shotgun metagenomic sequencing. Our results suggest that inferred community composition was dependent on inherent specimen properties as well as DNA extraction method. We further show that bead beating or enzymatic treatment can increase the extraction of DNA from Gram-positive bacteria. Final DNA quantities could be increased by isolating DNA from a larger volume of cell lysate than that in standard protocols. Based on this insight, we designed an improved DNA isolation procedure optimized for microbiome genomics that can be used for the three examined specimen types and potentially also for other biological specimens. A standard operating procedure is available from https://dx.doi.org/10.6084/m9.figshare.3475406. IMPORTANCE Sequencing-based analyses of microbiomes may lead to a breakthrough in our understanding of the microbial worlds associated with

  17. Comparison of DNA extraction methods for polymerase chain reaction amplification of guanaco (Lama guanicoe) fecal DNA samples.

    PubMed

    Espinosa, M I; Bertin, A; Squeo, F A; Cortés, A; Gouin, N

    2015-01-23

    Feces-based population genetic studies have become increasingly popular. However, polymerase chain reaction (PCR) amplification rates from fecal material vary depending on the species, populations, loci, and extraction protocols. Here, we assessed the PCR amplification success of three microsatellite markers and a segment of the mitochondrial control region of DNA extracted from field-collected feces of guanaco (Lama guanicoe) using two protocols - Qiagen DNA Stool Kit and 2 cetyltrimethylammonium bromide/phenol:chloroform:isoamyl alcohol (2CTAB/PCI) method. Chelex resin treatment to remove inhibitors was also tested. Our results show that the mitochondrial locus was the most difficult to amplify. PCR success rates improved for all markers after Chelex treatment of extracted DNA, and 2CTAB/PCI method (95.83%) appeared to perform slightly better than stool kit (91.67%) for the nuclear markers. Amplification success was significantly influenced by the extraction method, Chelex treatment, and locus (P < 0.001) but not by the freshness of the feces (fresh vs old, P = 0.17). The repeatability levels were high without Chelex treatment (> 0.89), but they decreased slightly after treatment for amplification of nuclear markers and markedly after treatment for amplification of the mitochondrial control region. Thus, we showed that Chelex treatment gives high PCR success, especially for nuclear markers, and adequate DNA extraction rates can be achieved from L. guanicoe feces even from non-fresh fecal material. Although not significant, 2CTAB/PCI method tended to provide higher successful amplification rates on a whole set of samples, suggesting that the method could be particularly useful when using small sample sizes.

  18. Use of buccal swabs for sampling DNA from nestling and adult birds

    USGS Publications Warehouse

    Handel, Colleen M.; Pajot, Lisa; Talbot, Sandra L.; Sage, George K.

    2006-01-01

    We evaluated the feasibility and efficiency of using swabs to collect buccal epithelial cells fromsmall (2‐ to 13‐g) birds as a source of DNA for genetic studies. We used commercially available buccal swab kits to collect samples from 42 adult and 39 nestling (4‐ to 8‐day‐old) black‐capped chickadees (Poecile atricapillus) and from6 4‐day‐old nestling boreal chickadees (P. hudsonica). We compared DNA from buccal epithelial samples to that fromblood samples from the same individuals. We extracted sufficient quantities of DNA for analysis from all buccalsamples, and samples remained viable even after being stored in original plastic sampling tubes at room temperature for up to 18 months. Yields were equivalent whether extracted using the proprietary quick‐extraction solution provided with buccal swab kits or using a salt‐extraction process with inexpensive reagents. Yields of DNA from buccal samples were consistently lower than those from blood samples, but quantities were sufficient for all analyses. Assignment of sex, based on DNA extracted from paired buccal and blood samples, was identical for all 87 birds. We found no difference in the genotypes obtained from buccal and blood samples for 12 individuals tested using 5 microsatellite loci and found perfect concordance in sequencing of an 823‐base‐pair segment within the control region of mitochondrial DNA for 7 individuals tested. Use of buccal swabs is highly recommended as a rapid, noninvasive technique for sampling avian genomic DNA, especially for extremely young altricial nestlings or small‐bodied adults, or for any birds for which blood sampling may be impossible or stressful.

  19. Forensic Analysis of Human DNA from Samples Contamined with Bioweapons Agents

    DTIC Science & Technology

    2011-10-01

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

  20. DNA-based determination of microbial biomass suitable for frozen and alkaline soil samples

    NASA Astrophysics Data System (ADS)

    Semenov, Mikhail; Blagodatskaya, Evgeniya; Kogut, Boris; Kuzyakov, Yakov

    2015-04-01

    Microbial biomass is a sensitive indicator of changes due to soil management, long before other basic soil measures such as Corg or Ntot. Improvement of methods for determination of microbial biomass still remains relevant, and these methods should be correctly applicable for the soil samples being in various state. This study was designed to demonstrate the applicability of DNA-based determination of microbial biomass under conditions when the common basic approaches, namely chloroform fumigation-extraction (CFE) and substrate-induced respiration (SIR), are restricted by certain soil properties, experimental designs or research needs, e.g. in frozen, alkaline or carbonaceous soils. We compared microbial biomass determined by CFE, SIR and by DNA approaches in the range of neutral and slightly alkaline Chernozem and alkaline Calcisol of semi-arid climate. The samples of natural and agricultural ecosystems were taken throughout the soil profile from long-term static field experiments in the European part of Russia. Extraction and subsequent quantification of dsDNA revealed a strong agreement with SIR and CFE when analyzing the microbial biomass content in soils with pH below 8. The conversion factors (FDNA) from dsDNA to SIR-Cmic (5.10) and CFE-Cmic (4.41) were obtained by testing a range of the soil samples down to 1.5 m depth and indicated a good reproducibility of DNA-based estimations. In alkaline soils (pH > 8), CO2 retention due to alkaline pH and exchange with carbonates resulted in a strong underestimation of soil microbial biomass by SIR or even in the absence of any CO2 emission, especially at low absolute values of microbial biomass in subsoil. Correction of CO2 efflux by theoretical retention pH-dependent factors caused overestimation of SIR-biomass. In alkaline conditions, DNA extraction proved to be a reliable alternative for microbial biomass determination. Moreover, the DNA-based approach can serve as an excellent alternative enabling correct

  1. A New Class of Quinoline-Based DNA Hypomethylating Agents Reactivates Tumor Suppressor Genes by Blocking DNA Methyltransferase 1 Activity and Inducing Its Degradation

    PubMed Central

    Datta, Jharna; Ghoshal, Kalpana; Denny, William A.; Gamage, Swarna A.; Brooke, Darby G.; Phiasivongsa, Pasit; Redkar, Sanjeev; Jacob, Samson T.

    2010-01-01

    Reactivation of silenced tumor suppressor genes by 5-azacytidine (Vidaza) and its congener 5-aza-2′-deoxycytidine (decitabine) has provided an alternate approach to cancer therapy. We have shown previously that these drugs selectively and rapidly induce degradation of the maintenance DNA methyltransferase (DNMT) 1 by a proteasomal pathway. Because the toxicity of these compounds is largely due to their incorporation into DNA, it is critical to explore novel, nonnucleoside compounds that can effectively reactivate the silenced genes. Here, we report that a quinoline-based compound, designated SGI-1027, inhibits the activity of DNMT1, DNMT3A, and DNMT3B as well M. SssI with comparable IC50 (6–13 µ mol/L) by competing with S-adenosylmethionine in the methylation reaction. Treatment of different cancer cell lines with SGI-1027 resulted in selective degradation of DNMT1 with minimal or no effects on DNMT3A and DNMT3B. At a concentration of 2.5 to 5 µmol/L (similar to that of decitabine), complete degradation of DNMT1 protein was achieved within 24 h without significantly affecting its mRNA level. MG132 blocked SGI-1027–induced depletion of DNMT1, indicating the involvement of proteasomal pathway. Prolonged treatment of RKO cells with SGI-1027 led to demethylation and reexpression of the silenced tumor suppressor genes P16, MLH1, and TIMP3. Further, this compound did not exhibit significant toxicity in a rat hepatoma (H4IIE) cell line. This study provides a novel class of DNA hypomethylating agents that have the potential for use in epigenetic cancer therapy. PMID:19417133

  2. Antibiotic resistance genes in the bacteriophage DNA fraction of environmental samples.

    PubMed

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

    2011-03-03

    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, β-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to β-lactam antibiotics is conferred by β-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 β-lactam antibiotics, namely two β-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.

  3. Antibiotic Resistance Genes in the Bacteriophage DNA Fraction of Environmental Samples

    PubMed Central

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

    2011-01-01

    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, β-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to β-lactam antibiotics is conferred by β-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 β-lactam antibiotics, namely two β-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. PMID:21390233

  4. Sequence studies on mouse L-cell satellite DNA by base-specific degradation with T4 endonuclease IV.

    PubMed

    Harbers, K; Spencer, J H

    1978-10-24

    The base sequence of mouse L-cell satellite DNA was investigated by degradation of the two separated complementary strands with the base specific enzyme, T4 endonuclease IV. Digestion of the heavy strand DNA released a limited number of oligonucleotides which were separated by ionophoresis/homochromatography, isolated, and sequenced by the 'wandering spot' method. The light strand DNA was resistant to digestion with T4 endonuclease IV and no detectable amounts of oligonucleotides were released. The oligonucleotides obtained from the heavy strand were related in sequence, indicating that mouse satellite DNA derived from a short tandemly repeated sequence. The sequence of part of the original repeat unit is proposed to be C-A-T-T-T-T-T-C. Five major oligonucleotides were identified, all of which differ from the proposed original sequence by single base changes. The five major oligonucleotides occur with about equal frequency and together comprise approximately 50% of the oligonucleotides released by T4 endonuclease IV from the heavy strand DNA. In addition to the five major oligonucleotides, several oligonucleotides were found to occur in lesser amounts. Since these oligonucleotides are related to the major oligonucleotides, it is likely that they have arisen from them by mutation.

  5. E2F1 interactions with hHR23A inhibit its degradation and promote DNA repair

    PubMed Central

    Singh, Randeep K.; Dagnino, Lina

    2016-01-01

    Nucleotide excision repair (NER) is a major mechanism for removal of DNA lesions induced by exposure to UV radiation in the epidermis. Recognition of damaged DNA sites is the initial step in their repair, and requires multiprotein complexes that contain XPC and hHR23 proteins, or their orthologues. A variety of transcription factors are also involved in NER, including E2F1. In epidermal keratinocytes, UV exposure induces E2F1 phosphorylation, which allows it to recruit various NER factors to sites of DNA damage. However, the relationship between E2F1 and hHR23 proteins vis-à-vis NER has remained unexplored. We now show that E2F1 and hHR23 proteins can interact, and this interaction stabilizes E2F1, inhibiting its proteasomal degradation. Reciprocally, E2F1 regulates hHR23A subcellular localization, recruiting it to sites of DNA photodamage. As a result, E2F1 and hHR23A enhance DNA repair following exposure to UV radiation, contributing to genomic stability in the epidermis. PMID:27028861

  6. Statistical approaches to account for false-positive errors in environmental DNA samples.

    PubMed

    Lahoz-Monfort, José J; Guillera-Arroita, Gurutzeta; Tingley, Reid

    2016-05-01

    Environmental DNA (eDNA) sampling is prone to both false-positive and false-negative errors. We review statistical methods to account for such errors in the analysis of eDNA data and use simulations to compare the performance of different modelling approaches. Our simulations illustrate that even low false-positive rates can produce biased estimates of occupancy and detectability. We further show that removing or classifying single PCR detections in an ad hoc manner under the suspicion that such records represent false positives, as sometimes advocated in the eDNA literature, also results in biased estimation of occupancy, detectability and false-positive rates. We advocate alternative approaches to account for false-positive errors that rely on prior information, or the collection of ancillary detection data at a subset of sites using a sampling method that is not prone to false-positive errors. We illustrate the advantages of these approaches over ad hoc classifications of detections and provide practical advice and code for fitting these models in maximum likelihood and Bayesian frameworks. Given the severe bias induced by false-negative and false-positive errors, the methods presented here should be more routinely adopted in eDNA studies.

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

    PubMed

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

    2014-08-01

    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.

  8. 16S ribosomal DNA clone libraries to reveal bacterial diversity in anaerobic reactor-degraded tetrabromobisphenol A.

    PubMed

    Peng, Xingxing; Zhang, Zaili; Zhao, Ziling; Jia, Xiaoshan

    2012-05-01

    Microorganisms able to rapidly degrade tetrabromobisphenol A (TBBPA) were domesticated in an anaerobic reactor and added to gradually increased concentrations of TBBPA. After 240 days of domestication, the degradation rate reached 96.0% in cultivated batch experiments lasting 20 days. The optimum cultivating temperature and pH were 30°C and 7.0. The bacterial community's composition and diversity in the reactor was studied by comparative analysis with 16S ribosomal DNA clone libraries. Amplified rDNA restriction analysis of 200 clones from the library indicate that the rDNA richness was high (Coverage C 99.5%) and that evenness was not high (Shannon-Weaver index 2.42). Phylogenetic analysis of 63 bacterial sequences from the reactor libraries demonstrated the presence of Betaproteobacteria (33.1%), Gammaproteobacteria (18.7%), Bacteroidetes (13.9%), Firmicutes (11.4%), Chloroflexi (3.6%), Actinobacteria (0.6%), the candidate division TM7 (4.2%) and other unknown, uncultured bacterial groups (14.5%). Comamonas, Achromobacter, Pseudomonas and Flavobacterium were the dominant types.

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

    NASA Technical Reports Server (NTRS)

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

    1973-01-01

    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.

  10. Antibiotic resistance genes in the bacteriophage DNA fraction of human fecal samples.

    PubMed

    Quirós, Pablo; Colomer-Lluch, Marta; Martínez-Castillo, Alexandre; Miró, Elisenda; Argente, Marc; Jofre, Juan; Navarro, Ferran; Muniesa, Maite

    2014-01-01

    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.

  11. Degradation of vanillic acid and production of guaiacol by microorganisms isolated from cork samples.

    PubMed

    Alvarez-Rodríguez, María Luisa; Belloch, Carmela; Villa, Mercedes; Uruburu, Federico; Larriba, Germán; Coque, Juan José R

    2003-03-14

    The presence of guaiacol in cork stoppers is responsible for some cases of cork taint causing unpleasant alterations to wine. We have performed a characterization of the cork-associated microbiota by isolating 55 different microorganisms: eight yeast, 14 filamentous fungi or molds, 13 actinomycetes and 20 non-filamentous bacteria. A screening for degradation of vanillic acid and guaiacol production showed that none of the filamentous fungi could achieve any of these processes. By contrast, five of the eight yeast strains isolated were able to degrade vanillic acid, although it was not converted to guaiacol. Guaiacol production was only detected in four bacterial strains: one isolate of Bacillus subtilis and three actinomycetes, Streptomyces sp. A3, Streptomyces sp. A5 and Streptomyces sp. A13, were able to accumulate this compound in both liquid media and cultures over cork. These results suggest that guaiacol-mediated cork taint should be attributed to the degradative action of vanillic acid by bacterial strains growing on cork.

  12. 16S rDNA-based probes for two polycyclic aromatic hydrocarbon (PAH)-degrading soil Mycobacteria

    SciTech Connect

    Govindaswami, M.; Feldhake, D.J.; Loper, J.C.

    1994-12-31

    PAHs are a class of widespread pollutants, some of which have been shown to be genotoxic, hence the fate of these compounds in the environment is of considerable interest. Research on the biodegradation of 4 and 5 ring PAHs has been limited by the general lack of microbial isolates or consortia which can completely degrade these toxicants. Heitkamp and Cerniglia have described an oxidative soil Mycobacterium-strain PYR-1 that metabolizes pyrene and fluoranthene more rapidly than the 2 and 3 ring naphthalene and phenanthrene; although some metabolites of benzo-(a)-pyrene (BaP) were detected, no mineralization of BaP was observed. In 1991 Grosser et al. reported the isolation of a Mycobacterium sp. which mineralizes pyrene and also causing some mineralization of BaP. Their study describes a comparative analysis of these two strains, which show very similar colony morphology, growth rate and yellow-orange pigmentation. Genetic differences were shown by DNA amplification fingerprinting (DAF) using two arbitrary GC-rich octanucleotide primers, and by sequence comparison of PCR amplified 16S rDNA, although both strains show similarity closest to that of the genus Mycobacteria. These 16S rDNA sequences are in use for the construction of strain-specific DNA probes to monitor the presence, survival and growth of these isolates in PAH-contaminated soils in studies of biodegradation.

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

    SciTech Connect

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

    2006-10-01

    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.

  14. DNA recognition by peptide nucleic acid-modified PCFs: from models to real samples

    NASA Astrophysics Data System (ADS)

    Selleri, S.; Coscelli, E.; Poli, F.; Passaro, D.; Cucinotta, A.; Lantano, C.; Corradini, R.; Marchelli, R.

    2010-04-01

    The increased concern, emerged in the last few years, on food products safety has stimulated the research on new techniques for traceability of raw food materials. DNA analysis is one of the most powerful tools for the certification of food quality, and it is presently performed through the polymerase chain reaction technique. Photonic crystal fibers, due to the presence of an array of air holes running along their length, can be exploited for performing DNA recognition by derivatizing hole surfaces and checking hybridization of complementary nucledotide chains in the sample. In this paper the application of a suspended core photonic crystal fiber in the recognition of DNA sequences is discussed. The fiber is characterized in terms of electromagnetic properties by means of a full-vector modal solver based on the finite element method. Then, the performances of the fiber in the recognition of mall synthetic oligonucleotides are discussed, together with a test of the possibility to extend this recognition to samples of DNA of applicative interest, such as olive leaves.

  15. I Environmental DNA sampling is more sensitive than a traditional survey technique for detecting an aquatic invader.

    PubMed

    Smart, Adam S; Tingley, Reid; Weeks, Andrew R; van Rooyen, Anthony R; McCarthy, Michael A

    2015-10-01

    Effective management of alien species requires detecting populations in the early stages of invasion. Environmental DNA (eDNA) sampling can detect aquatic species at relatively low densities, but few studies have directly compared detection probabilities of eDNA sampling with those of traditional sampling methods. We compare the ability of a traditional sampling technique (bottle trapping) and eDNA to detect a recently established invader, the smooth newt Lissotriton vulgaris vulgaris, at seven field sites in Melbourne, Australia. Over a four-month period, per-trap detection probabilities ranged from 0.01 to 0.26 among sites where L. v. vulgaris was detected, whereas per-sample eDNA estimates were much higher (0.29-1.0). Detection probabilities of both methods varied temporally (across days and months), but temporal variation appeared to be uncorrelated between methods. Only estimates of spatial variation were strongly correlated across the two sampling techniques. Environmental variables (water depth, rainfall, ambient temperature) were not clearly correlated with detection probabilities estimated via trapping, whereas eDNA detection probabilities were negatively correlated with water depth, possibly reflecting higher eDNA concentrations at lower water levels. Our findings demonstrate that eDNA sampling can be an order of magnitude more sensitive than traditional methods, and illustrate that traditional- and eDNA-based surveys can provide independent information on species distributions when occupancy surveys are conducted over short timescales.

  16. Developmental validation of the DNAscan™ Rapid DNA Analysis™ instrument and expert system for reference sample processing.

    PubMed

    Della Manna, Angelo; Nye, Jeffrey V; Carney, Christopher; Hammons, Jennifer S; Mann, Michael; Al Shamali, Farida; Vallone, Peter M; Romsos, Erica L; Marne, Beth Ann; Tan, Eugene; Turingan, Rosemary S; Hogan, Catherine; Selden, Richard F; French, Julie L

    2016-11-01

    Since the implementation of forensic DNA typing in labs more than 20 years ago, the analysis procedures and data interpretation have always been conducted in a laboratory by highly trained and qualified scientific personnel. Rapid DNA technology has the potential to expand testing capabilities within forensic laboratories and to allow forensic STR analysis to be performed outside the physical boundaries of the traditional laboratory. The developmental validation of the DNAscan/ANDE Rapid DNA Analysis System was completed using a BioChipSet™ Cassette consumable designed for high DNA content samples, such as single source buccal swabs. A total of eight laboratories participated in the testing which totaled over 2300 swabs, and included nearly 1400 unique individuals. The goal of this extensive study was to obtain, document, analyze, and assess DNAscan and its internal Expert System to reliably genotype reference samples in a manner compliant with the FBI's Quality Assurance Standards (QAS) and the NDIS Operational Procedures. The DNAscan System provided high quality, concordant results for reference buccal swabs, including automated data analysis with an integrated Expert System. Seven external laboratories and NetBio, the developer of the technology, participated in the validation testing demonstrating the reproducibility and reliability of the system and its successful use in a variety of settings by numerous operators. The DNAscan System demonstrated limited cross reactivity with other species, was resilient in the presence of numerous inhibitors, and provided reproducible results for both buccal and purified DNA samples with sensitivity at a level appropriate for buccal swabs. The precision and resolution of the system met industry standards for detection of micro-variants and displayed single base resolution. PCR-based studies provided confidence that the system was robust and that the amplification reaction had been optimized to provide high quality results

  17. Electrochemical DNA hybridization sensors applied to real and complex biological samples.

    PubMed

    Tosar, J P; Brañas, G; Laíz, J

    2010-12-15

    DNA hybridization biosensors, also known as genosensors, are analytical devices for the detection of specific DNA "target" sequences in solution, upon hybridization of the targets with complementary "probes" immobilized on a solid substrate. Electrochemical genosensors hold great promise to serve as devices suitable for point-of-care diagnostics and multiplexed platforms for fast, simple and inexpensive nucleic acids analysis. Although a lot of progress has been made in the past few years, the performance of genosensors in complex biological samples has been assayed in only a small fraction of published research articles. This review covers such a group of reports, from the year 2000 onwards. Special attention is played in the nature and complexity of the samples and in the way matrix effects were treated and specificity controls were performed.

  18. Horizontal transfer of short and degraded DNA has evolutionary implications for microbes and eukaryotic sexual reproduction

    PubMed Central

    Overballe-Petersen, Søren; Willerslev, Eske

    2014-01-01

    Horizontal gene transfer in the form of long DNA fragments has changed our view of bacterial evolution. Recently, we discovered that such processes may also occur with the massive amounts of short and damaged DNA in the environment, and even with truly ancient DNA. Although it presently remains unclear how often it takes place in nature, horizontal gene transfer of short and damaged DNA opens up the possibility for genetic exchange across distinct species in both time and space. In this essay, we speculate on the potential evolutionary consequences of this phenomenon. We argue that it may challenge basic assumptions in evolutionary theory; that it may have distant origins in life's history; and that horizontal gene transfer should be viewed as an evolutionary strategy not only preceding but causally underpinning the evolution of sexual reproduction. PMID:25143190

  19. Characterization of Markers of the Progression of Human Parvovirus B19 Infection in Virus DNA-Positive Plasma Samples

    PubMed Central

    Bonjoch, Xavier; Obispo, Francesc; Alemany, Cristina; Pacha, Ana; Rodríguez, Esteban; Xairó, Dolors

    2015-01-01

    Summary Background Accurate characterization of the infection stage in parvovirus B19(B19V)-positive plasma donations would help establish the donation deferral period to contribute to a safe fractionation pool of plasma. Methods Viral DNA load of 74 B19V DNA-positive plasma samples from whole blood donations was determined by titration using nucleic acid testing. Markers of cellular (neopterin) and humoral (B19V-specific IgM and IgG) immune response were determined by ELISA in 32 B19V DNA-positive samples and in 13 B19V DNA-negative samples. The infection progression profile was estimated according to B19V DNA load and the presence of immune response markers. Results B19V DNA load in the 74 samples was 106-1013 IU/ml. The distribution of 14 out of 32 selected B19V DNA-positive samples plus 2 B19V DNA-negative samples with no immune response marker followed along an upward curve according to B19V DNA load. After the peak, the distribution of 18 immune marker-positive samples followed along a downward curve according to their B19V DNA load and was grouped as follows: neopterin (n = 4), neopterin+ IgM (n = 8), neopterin + IgM + IgG (n = 3), IgM + IgG (n = 2), IgM (n = 1). There were 11 B19V DNA-negative IgG-positive samples. Conclusion This study of B19V-DNA load and levels of neopterin, IgM, and IgG allows for reliable characterization and distribution into the different stages of B19V infection. PMID:26557815

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

    PubMed

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

    2014-05-01

    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.

  1. Bisulfite-Based DNA Methylation Analysis from Recent and Archived Formalin-Fixed, Paraffin Embedded Colorectal Tissue Samples.

    PubMed

    Kalmár, Alexandra; Péterfia, Bálint; Hollósi, Péter; Wichmann, Barnabás; Bodor, András; Patai, Árpád V; Schöller, Andrea; Krenács, Tibor; Tulassay, Zsolt; Molnár, Béla

    2015-09-01

    We aimed to test the applicability of formalin-fixed and paraffin-embedded (FFPE) tissue samples for gene specific DNA methylation analysis after using two commercially available DNA isolation kits. Genomic DNA was isolated from 5 colorectal adenocarcinomas and 5 normal adjacent tissues from "recent", collected within 6 months, and "archived", collected more than 5 years ago, FFPE tissues using either High Pure FFPET DNA Isolation kit or QIAamp DNA FFPE Tissue kit. DNA methylation analysis of MAL, SFRP1 and SFRP2 genes, known to be hypermethylated in CRC, was performed using methylation-sensitive high resolution melting (MS-HRM) analysis and sequencing. QIAamp (Q) method resulted in slightly higher recovery in archived (HP: 1.22 ± 3.18 μg DNA; Q: 3.00 ± 4.04 μg DNA) and significantly (p < 0.05) higher recovery in recent samples compared to High Pure method (HP) (HP: 4.10 ± 2.91 μg DNA; Q: 11.51 ± 7.50 μg DNA). Both OD260/280 and OD260/230 ratios were lower, but still high in the High Pure isolated archived and recent samples compared to those isolated with QIAamp. Identical DNA methylation patterns were detected for all 3 genes tested by MS-HRM with both isolation kits in the recent group. However, despite of higher DNA recovery in QIAamp slightly more reproducible methylation results were obtained from High Pure isolated archived samples. Sequencing confirmed DNA hypermethylation in CRCs. In conclusion, reproducible DNA methylation patterns were obtained from recent samples using both isolation kits. However, long term storage may affect the reliability of the results leading to moderate differences between the efficiency of isolation kits.

  2. Unraveling a mechanism of honey antibacterial action: polyphenol/H₂O₂-induced oxidative effect on bacterial cell growth and on DNA degradation.

    PubMed

    Brudzynski, Katrina; Abubaker, Kamal; Miotto, Danielle

    2012-07-15

    Several compounds with antibacterial activities were identified in honey however, a mechanism by which they lead to bacterial growth inhibition and bacterial death remains still unknown. We recently found that honeys possess DNA degrading activity mediated by honey hydrogen peroxide and an unknown honey component(s). Here we provide evidence that active honeys (MIC90 of 6.25-12.5% v/v) possessed significantly higher levels of phenolics (p<0.02) of higher radical scavenging activities (p<0.005) than honeys of average activity. Removal of H2O2 by catalase eliminated bacteriostatic activities caused by both phenolics and H2O2 suggesting that the growth inhibition resulted from the coupling chemistry between these compounds. Both phenolics and H2O2 were involved in DNA degradation by honeys. Treatment of plasmid DNA with H2O2 alone did not affect the DNA integrity but H2O2 removal from honey by catalase prevented DNA degradation. Polyphenols extracted from honeys degraded plasmid DNA in the presence of H2O2 and Cu(II) in the Fenton-type reaction. The extent of DNA degradation was inversely related to the polyphenol concentration in this system as well as in honeys. At low content, honey polyphenols exerted pro-oxidant activity damaging to DNA. In conclusion, honey phenolics with pro-oxidant activities were necessary intermediates that conferred oxidative action of H2O2. Phenolic/H2O2-induced oxidative stress constituted the mechanism of honey bacteriostatic and DNA damaging activities.

  3. Vertebrate DNA in Fecal Samples from Bonobos and Gorillas: Evidence for Meat Consumption or Artefact?

    PubMed Central

    Hofreiter, Michael; Kreuz, Eva; Eriksson, Jonas; Schubert, Grit; Hohmann, Gottfried

    2010-01-01

    Background Deciphering the behavioral repertoire of great apes is a challenge for several reasons. First, due to their elusive behavior in dense forest environments, great ape populations are often difficult to observe. Second, members of the genus Pan are known to display a great variety in their behavioral repertoire; thus, observations from one population are not necessarily representative for other populations. For example, bonobos (Pan paniscus) are generally believed to consume almost no vertebrate prey. However, recent observations show that at least some bonobo populations may consume vertebrate prey more commonly than previously believed. We investigated the extent of their meat consumption using PCR amplification of vertebrate mitochondrial DNA (mtDNA) segments from DNA extracted from bonobo feces. As a control we also attempted PCR amplifications from gorilla feces, a species assumed to be strictly herbivorous. Principal Findings We found evidence for consumption of a variety of mammalian species in about 16% of the samples investigated. Moreover, 40% of the positive DNA amplifications originated from arboreal monkeys. However, we also found duiker and monkey mtDNA in the gorilla feces, albeit in somewhat lower percentages. Notably, the DNA sequences isolated from the two ape species fit best to the species living in the respective regions. This result suggests that the sequences are of regional origin and do not represent laboratory contaminants. Conclusions Our results allow at least three possible and mutually not exclusive conclusions. First, all results may represent contamination of the feces by vertebrate DNA from the local environment. Thus, studies investigating a species' diet from feces DNA may be unreliable due to the low copy number of DNA originating from diet items. Second, there is some inherent difference between the bonobo and gorilla feces, with only the later ones being contaminated. Third, similar to bonobos, for which the

  4. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

    NASA Astrophysics Data System (ADS)

    Ali, M. E.; Hashim, U.; Mustafa, S.; Che Man, Y. B.; Yusop, M. H. M.; Bari, M. F.; Islam, Kh N.; Hasan, M. F.

    2011-05-01

    We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 °C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 µg ml - 1 swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

  5. Association of telomere length and mitochondrial DNA copy number in a community sample of healthy adults.

    PubMed

    Tyrka, Audrey R; Carpenter, Linda L; Kao, Hung-Teh; Porton, Barbara; Philip, Noah S; Ridout, Samuel J; Ridout, Kathryn K; Price, Lawrence H

    2015-06-01

    Cellular aging plays a role in longevity and senescence, and has been implicated in medical and psychiatric conditions, including heart disease, cancer, major depression and posttraumatic stress disorder. Telomere shortening and mitochondrial dysfunction are thought to be central to the cellular aging process. The present study examined the association between mitochondrial DNA (mtDNA) copy number and telomere length in a sample of medically healthy adults. Participants (total n=392) were divided into 4 groups based on the presence or absence of early life adversity and lifetime psychopathology: No Adversity/No Disorder, n=136; Adversity/No Disorder, n=91; No Adversity/Disorder, n=46; Adversity/Disorder, n=119. Telomere length and mtDNA copy number were measured using quantitative polymerase chain reaction. There was a positive correlation between mtDNA and telomere length in the entire sample (r=0.120, p<0.001) and in each of the four groups of participants (No Adversity/No Disorder, r=0.291, p=0.001; Adversity/No Disorder r=0.279, p=0.007; No Adversity/Disorder r=0.449, p=0.002; Adversity/Disorder, r=0.558, p<0.001). These correlations remained significant when controlling for age, smoking, and body mass index and establish an association between mtDNA and telomere length in a large group of women and men both with and without early adversity and psychopathology, suggesting co-regulation of telomeres and mitochondrial function. The mechanisms underlying this association may be important in the pathophysiology of age-related medical conditions, such as heart disease and cancer, as well as for stress-associated psychiatric disorders.

  6. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples.

    PubMed

    Ali, M E; Hashim, U; Mustafa, S; Man, Y B Che; Yusop, M H M; Bari, M F; Islam, Kh N; Hasan, M F

    2011-05-13

    We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 °C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 µg ml(-1) swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

  7. DNA sampling from eggshell swabbing is widely applicable in wild bird populations as demonstrated in 23 species.

    PubMed

    Martín-Gálvez, David; Peralta-Sánchez, Juan M; Dawson, Deborah A; Martín-Platero, Antonio M; Martínez-Bueno, Manuel; Burke, Terry; Soler, Juan J

    2011-05-01

    There is increasing interest in noninvasive DNA sampling techniques. In birds, there are several methods proposed for sampling DNA, and of these, the use of eggshell swabbing is potentially applicable to a wide range of species. We estimated the effectiveness of this method in the wild by sampling the eggs of 23 bird species. Sampling of eggs was performed twice per nest, soon after the clutch was laid and again at the end of egg incubation. We genotyped DNA samples using a set of five conserved microsatellite markers, which included a Z-linked locus and a sex-typing marker. We successfully collected avian DNA from the eggs of all species tested and from 88.48% of the samples. In most of the cases, the DNA concentration was low (ca. 10 ng/μL). The number of microsatellite loci amplified per sample (0-5) was used as a measure of the genotyping success of the sample. On average, we genotyped 3.01 ± 0.12 loci per sample (mean ± SE), and time of sampling did not seem to have an effect; however, genotyping success differed among species and was greater in those species that used feather material for lining their nest cups. We also checked for the occurrence of possible genotyping errors derived from using samples with very low DNA quantities (i.e. allelic dropout or false alleles) and for DNA contamination from individuals other than the mother, which appeared at a moderate rate (in 44% of the PCR replicates and in 17.36% of samples, respectively). Additionally, we investigated whether the DNA on eggshells corresponded to maternal DNA by comparing the genotypes obtained from the eggshells to those obtained from blood samples of all the nestlings for six nests of magpies. In five of the six magpie nests, we found evidence that the swab genotypes were a mixture of genotypes from both parents and this finding was independent of the time of incubation. Thus, our results broadly confirm that the swabbing of eggshells can be used as a noninvasive method for obtaining DNA

  8. To Clone or Not To Clone: Method Analysis for Retrieving Consensus Sequences In Ancient DNA Samples

    PubMed Central

    Winters, Misa; Barta, Jodi Lynn; Monroe, Cara; Kemp, Brian M.

    2011-01-01

    The challenges associated with the retrieval and authentication of ancient DNA (aDNA) evidence are principally due to post-mortem damage which makes ancient samples particularly prone to contamination from “modern” DNA sources. The necessity for authentication of results has led many aDNA researchers to adopt methods considered to be “gold standards” in the field, including cloning aDNA amplicons as opposed to directly sequencing them. However, no standardized protocol has emerged regarding the necessary number of clones to sequence, how a consensus sequence is most appropriately derived, or how results should be reported in the literature. In addition, there has been no systematic demonstration of the degree to which direct sequences are affected by damage or whether direct sequencing would provide disparate results from a consensus of clones. To address this issue, a comparative study was designed to examine both cloned and direct sequences amplified from ∼3,500 year-old ancient northern fur seal DNA extracts. Majority rules and the Consensus Confidence Program were used to generate consensus sequences for each individual from the cloned sequences, which exhibited damage at 31 of 139 base pairs across all clones. In no instance did the consensus of clones differ from the direct sequence. This study demonstrates that, when appropriate, cloning need not be the default method, but instead, should be used as a measure of authentication on a case-by-case basis, especially when this practice adds time and cost to studies where it may be superfluous. PMID:21738625

  9. Comparison of DNA extraction methods from small samples of newborn screening cards suitable for retrospective perinatal viral research.

    PubMed

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

    2011-04-01

    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/μl. The limit of detection was 10 viral genome copies in a 50-μl 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.

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

    NASA Technical Reports Server (NTRS)

    1978-01-01

    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.

  11. Sample selection algorithm to improve quality of genotyping from plasma-derived DNA: to separate the wheat from the chaff.

    PubMed

    Schoenborn, Veit; Gohlke, Henning; Heid, Iris M; Illig, Thomas; Utermann, Gerd; Kronenberg, Florian

    2007-11-01

    Plasma and serum samples were often the only biological material collected for earlier epidemiological studies. These studies have a huge informative content, especially due to their long follow-up and would be an invaluable treasure for genetic investigations. However, often no banked DNA is available. To use the small amounts of DNA present in plasma, in a first step, we applied magnetic bead technology to extract this DNA, followed by a whole-genome amplification (WGA) using phi29-polymerase. We assembled 88 sample pairs, each consisting of WGA plasma DNA and the corresponding whole-blood DNA. We genotyped nine highly polymorphic short tandem repeats (STRs) and 23 SNPs in both DNA sources. The average within-pair discordance was 3.8% for SNPs and 15.9% for STR genotypes, respectively. We developed an algorithm based on one-half of the sample pairs and validated on the other one-half to identify the samples with high WGA plasma DNA quality to assure low genotyping error and to exclude plasma DNA samples with insufficient quality: excluding samples showing homozygosity at five or more of the nine STR loci yielded exclusion of 22.7% of all samples and decreased average discordance for STR and SNP markers to 3.92% and 0.63%, respectively. For SNPs, this is very close to the error observed for genomic DNA in many laboratories. Our workflow and sample selection algorithm offers new opportunities to recover reliable DNA from stored plasma material. This algorithm is superior to testing the amount of input DNA.

  12. DNA adduct analysis and histopathological biomarkers in European flounder (Platichthys flesus) sampled from UK estuaries.

    PubMed

    Lyons, B P; Stentiford, G D; Green, M; Bignell, J; Bateman, K; Feist, S W; Goodsir, F; Reynolds, W J; Thain, J E

    2004-08-18

    The presence of genotoxic and potentially carcinogenic chemical contaminants in the estuarine and coastal marine environment is well documented. In this study, European flounder (Platichthys flesus) sampled from eight UK estuaries were analysed for hepatic DNA adducts, using the 32P-postlabelling assay and liver histopathology as part of an on going survey to establish the health status of UK estuaries. Fish were collected from the estuaries Tyne, Mersey, Thames, Alde (reference site), Belfast, Forth, Clyde and Southampton. At the majority of contaminated sites (Southampton, Thames, Clyde, Tyne and Mersey) the predominant DNA adduct profile consisted of diagonal radioactive zones (DRZs). In contrast, flounder collected from the Forth, Alde and Belfast lacked DRZs with only background levels of DNA damage being observed. Statistically significant differences were observed between several of the sites sampled with the hepatic DNA adduct levels detected in flounder from Southampton, Thames and Clyde statistically elevated (P < 0.05) over those levels detected at the Tyne (Southampton and Thames only), Forth, Alde and Belfast. Histological analysis of these samples revealed a range of lesions including foci of cellular alteration, hepatocellular fibrillar inclusions, nuclear pleomorphisms along with non-toxicopathic changes/alterations, such as those associated with cell turnover (apoptosis, necrosis, regeneration) and immune-related functions (melanomacrophage aggregates, inflammation). Although it is difficult to associate higher prevalence of these lesion types with specific sites, generally, the lowest prevalence was seen in flounder captured from the Alde estuary, with higher prevalence (particularly of melanomacrophage aggregates, inflammation and necrotic foci) seen in fish from the contaminated sites.

  13. 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...

  14. Multiplex short tandem repeat amplification of low template DNA samples with the addition of proofreading enzymes.

    PubMed

    Davis, Carey P; Chelland, Lynzee A; Pavlova, Victoria R; Illescas, María J; Brown, Kelly L; Cruz, Tracey Dawson

    2011-05-01

    With <100 pg of template DNA, routine short tandem repeat (STR) analysis often fails, resulting in no or partial profiles and increased stochastic effects. To overcome this, some have investigated preamplification methods that include the addition of proofreading enzymes to the PCR cocktail. This project sought to determine whether adding proofreading polymerases directly in the STR amplification mixture would improve the reaction when little template DNA is available. Platinum Taq High Fidelity and GeneAmp High Fidelity were tested in Profiler Plus™ STR reactions alone and in combination with AmpliTaq(®) Gold. All reactions included the additional step of a post-PCR purification step. With both pristine low template DNA and casework samples, the addition of these polymerases resulted in comparable or no improvement in the STR amplification signal. Further, stochastic effects and artifacts were observed equally across all enzyme conditions. Based on these studies, the addition of these proofreading enzymes to a multiplex STR amplification is not recommended for low template DNA work.

  15. Objective data on DNA success rates can aid the selection process of crime samples for analysis by rapid mobile DNA technologies.

    PubMed

    Mapes, A A; Kloosterman, A D; Poot, C J de; van Marion, V

    2016-07-01

    Mobile Rapid-DNA devices have recently become available on the market. These devices can perform DNA analyses within 90min with an easy 'sample in-answer out' system, with the option of performing comparisons with a DNA database or reference profile. However, these fast mobile systems cannot yet compete with the sensitivity of the standard laboratory analysis. For the future this implies that Scene of Crime Officers (SoCOs) need to decide on whether to analyse a crime sample with a Rapid-DNA device and to get results within 2h or to secure and analyse the sample at the laboratory with a much longer throughput time but with higher sensitivity. This study provides SoCOs with evidence-based information on DNA success rates, which can improve their decisions at the crime scene on whether or not to use a Rapid-DNA device. Crime samples with a high success rate in the laboratory will also have the highest potential for Rapid-DNA analysis. These include samples from e.g. headwear, cigarette ends, articles of clothing, bloodstains, and drinking items.

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

    PubMed Central

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

    2009-01-01

    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

  17. Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.

    PubMed

    Henderson, Gemma; Cox, Faith; Kittelmann, Sandra; Miri, Vahideh Heidarian; Zethof, Michael; Noel, Samantha J; Waghorn, Garry C; Janssen, Peter H

    2013-01-01

    Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data

  18. CDC-48/p97 coordinates CDT-1 degradation with GINS chromatin dissociation to ensure faithful DNA replication.

    PubMed

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

    2011-10-07

    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-48(UFD-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.

  19. SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage

    PubMed Central

    Hong, Xuehui; Liu, Wenyu; Song, Ruipeng; Shah, Jamie J.; Feng, Xing; Tsang, Chi Kwan; Morgan, Katherine M.; Bunting, Samuel F.; Inuzuka, Hiroyuki; Zheng, X. F. Steven; Shen, Zhiyuan; Sabaawy, Hatem E.; Liu, LianXin; Pine, Sharon R.

    2016-01-01

    SOX9 encodes a transcription factor that governs cell fate specification throughout development and tissue homeostasis. Elevated SOX9 is implicated in the genesis and progression of human tumors by increasing cell proliferation and epithelial-mesenchymal transition. We found that in response to UV irradiation or genotoxic chemotherapeutics, SOX9 is actively degraded in various cancer types and in normal epithelial cells, through a pathway independent of p53, ATM, ATR and DNA-PK. SOX9 is phosphorylated by GSK3β, facilitating the binding of SOX9 to the F-box protein FBW7α, an E3 ligase that functions in the DNA damage response pathway. The binding of FBW7α to the SOX9 K2 domain at T236-T240 targets SOX9 for subsequent ubiquitination and proteasomal destruction. Exogenous overexpression of SOX9 after genotoxic stress increases cell survival. Our findings reveal a novel regulatory mechanism for SOX9 stability and uncover a unique function of SOX9 in the cellular response to DNA damage. This new mechanism underlying a FBW7-SOX9 axis in cancer could have implications in therapy resistance. PMID:27566146

  20. SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage.

    PubMed

    Hong, Xuehui; Liu, Wenyu; Song, Ruipeng; Shah, Jamie J; Feng, Xing; Tsang, Chi Kwan; Morgan, Katherine M; Bunting, Samuel F; Inuzuka, Hiroyuki; Zheng, X F Steven; Shen, Zhiyuan; Sabaawy, Hatem E; Liu, LianXin; Pine, Sharon R

    2016-10-14

    SOX9 encodes a transcription factor that governs cell fate specification throughout development and tissue homeostasis. Elevated SOX9 is implicated in the genesis and progression of human tumors by increasing cell proliferation and epithelial-mesenchymal transition. We found that in response to UV irradiation or genotoxic chemotherapeutics, SOX9 is actively degraded in various cancer types and in normal epithelial cells, through a pathway independent of p53, ATM, ATR and DNA-PK. SOX9 is phosphorylated by GSK3β, facilitating the binding of SOX9 to the F-box protein FBW7α, an E3 ligase that functions in the DNA damage response pathway. The binding of FBW7α to the SOX9 K2 domain at T236-T240 targets SOX9 for subsequent ubiquitination and proteasomal destruction. Exogenous overexpression of SOX9 after genotoxic stress increases cell survival. Our findings reveal a novel regulatory mechanism for SOX9 stability and uncover a unique function of SOX9 in the cellular response to DNA damage. This new mechanism underlying a FBW7-SOX9 axis in cancer could have implications in therapy resistance.

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

    PubMed

    Luz María Chacón, J; Lizeth Taylor, C; Carmen Valiente, A; Irene Alvarado, P; Ximena Cortés, B

    2012-10-01

    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.

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

    PubMed Central

    Luz María Chacón, J; Lizeth Taylor, C; Carmen Valiente, A; Irene Alvarado, P; Ximena Cortés, B

    2012-01-01

    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. PMID:24031959

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

    SciTech Connect

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

    1986-10-01

    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/sup 4/ physical particlels of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hydbridization stringency, /sup 32/P 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.

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

    SciTech Connect

    Tan, H.

    1999-03-31

    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%.

  5. Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples.

    PubMed

    Schmidt, M; Hoffmann, G; Wissler, M; Lemke, N; Müssig, A; Glimm, H; Williams, D A; Ragg, S; Hesemann, C U; von Kalle, C

    2001-05-01

    By identifying the sequence of retro- and lentiviral integration sites in peripheral blood leukocytes, the clonal composition and fate of genetically modified hematopoietic progenitor and stem cells could be mapped in vitro and in vivo. Previously available methods have been limited to the analysis of mono- or oligoclonal integration sites present in high copy numbers. Here, we perform characterization of multiple rare retroviral and lentiviral integration sites in highly complex DNA samples. The reliability of this method results from nontarget DNA removal via magnetic extension primer tag selection (EPTS) preceding solid-phase ligation-mediated PCR. EPTS/LM-PCR allowed the simultaneous direct genomic sequencing of multiple proviral LTR-flanking sequences of retro- and lentiviral vectors even if only 1 per 100 to 1000 cells contained the provirus. A primer walking "around" the integration locus demonstrated the adaptability of EPTS/LM-PCR to study unknown flanking DNA regions unrelated to proviruses. The technique is fast, inexpensive, and sensitive in minimal samples. It enables studies of retro- and lentiviral integration, viral vector tracking in gene therapy, insertional mutagenesis, transgene integration, and direct genomic sequencing that until now have been difficult or impossible to perform.

  6. Comparative analysis of three methods for HPV DNA detection in cervical samples.

    PubMed

    Michelli, Elvia; Téllez, Luis; Mendoza, José-Andrés; Jürgensen, Claudia; Muñoz, Maritza; Pérez, Saberio; Mosqueda, Noraida; Hernández, Erick; Noguera, María-Eugenia; Callejas, Diana; Correnti, María; Cavazza, María-Eugenia; Vielma, Silvana

    2011-12-01

    High risk HPV infection is considered to play a central role in cervical carcinogenesis. HPV DNA testing has shown to be a very useful tool for screening and following cervical infections. The aim of this study was to compare three methods for HPV DNA detection, along with cytology and colposcopy analysis. Cervical samples were collected from 100 sexually active women in Mérida, western Venezuela. HPV infection was screened using Hybrid-Capture 2 (HC2), L1-Nested-PCR and E6/E7-PCR assays. 40% of the samples (40/100) were HPV positive by at least one of the DNA detection methods. HC2 detected HPV in 12% specimens. L1- and E6/E7-PCRs showed 50% sensitivity and 77% specificity.The agreement rate between HC2 and both PCR assays was 65%. Kappa value showed moderate concordance between HC2 and both PCR methods (kappa=0.55; CI 95%). Also moderate concordance was seen when L1- and E6/E7-PCRs were compared (kappa=0.48; CI 95%). There was a significant association between the Schiller test and E6/E7-PCR (p=0.006) for HPV infection. An acceptable agreement between all three assays for HPV detection was observed. Nevertheless, different PCR formats need to be further analyzed in order to make the right choice of method for HPV testing.

  7. Microsatellite-based parentage analysis of Aedes aegypti (Diptera: Culicidae) using nonlethal DNA sampling.

    PubMed

    Wong, Jacklyn; Chu, Yui Yin; Stoddard, Steven T; Lee, Yoosook; Morrison, Amy C; Scott, Thomas W

    2012-01-01

    To track Aedes aegypti (L.) egg-laying behavior in the field in Iquitos, Peru, we developed methods for 1) sampling DNA from live mosquitoes and 2) high through-put parentage analysis using microsatellite markers. We were able to amplify DNA extracted from a single hind leg, but not from the pupal exuvia. Removal of a leg from teneral females caused no significant changes in female behavioral or life history traits (e.g., longevity, blood feeding frequency, fecundity, egg hatch rate, gonotrophic cycle length, or oviposition behavior). Using a panel of nine microsatellite markers and an exclusion-based software program, we matched offspring to parental pairs in 10 Ae. aegypti test families in which parents originated from natural development sites in Iquitos. By mating known individuals in the laboratory, retaining the male, sampling the female's DNA before release, and collecting offspring in the field, the technique we developed can be used to genotype large numbers of Ae. aegypti, reconstruct family relationships, and track the egg-laying behavior of individual Ae. aegypti in nature.

  8. Analysis and Optimization of Bulk DNA Sampling with Binary Scoring for Germplasm Characterization

    PubMed Central

    Reyes-Valdés, M. Humberto; Santacruz-Varela, Amalio; Martínez, Octavio; Simpson, June; Hayano-Kanashiro, Corina; Cortés-Romero, Celso

    2013-01-01

    The strategy of bulk DNA sampling has been a valuable method for studying large numbers of individuals through genetic markers. The application of this strategy for discrimination among germplasm sources was analyzed through information theory, considering the case of polymorphic alleles scored binarily for their presence or absence in DNA pools. We defined the informativeness of a set of marker loci in bulks as the mutual information between genotype and population identity, composed by two terms: diversity and noise. The first term is the entropy of bulk genotypes, whereas the noise term is measured through the conditional entropy of bulk genotypes given germplasm sources. Thus, optimizing marker information implies increasing diversity and reducing noise. Simple formulas were devised to estimate marker information per allele from a set of estimated allele frequencies across populations. As an example, they allowed optimization of bulk size for SSR genotyping in maize, from allele frequencies estimated in a sample of 56 maize populations. It was found that a sample of 30 plants from a random mating population is adequate for maize germplasm SSR characterization. We analyzed the use of divided bulks to overcome the allele dilution problem in DNA pools, and concluded that samples of 30 plants divided into three bulks of 10 plants are efficient to characterize maize germplasm sources through SSR with a good control of the dilution problem. We estimated the informativeness of 30 SSR loci from the estimated allele frequencies in maize populations, and found a wide variation of marker informativeness, which positively correlated with the number of alleles per locus. PMID:24260321

  9. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types.

    PubMed

    Lever, Mark A; Torti, Andrea; Eickenbusch, Philip; Michaud, Alexander B; Šantl-Temkiv, Tina; Jørgensen, Bo Barker

    2015-01-01

    A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world's oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals.

  10. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types

    PubMed Central

    Lever, Mark A.; Torti, Andrea; Eickenbusch, Philip; Michaud, Alexander B.; Šantl-Temkiv, Tina; Jørgensen, Bo Barker

    2015-01-01

    A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world's oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals. PMID:26042110

  11. Analysis of fingerprint samples, testing various conditions, for forensic DNA identification.

    PubMed

    Ostojic, Lana; Wurmbach, Elisa

    2017-01-01

    Fingerprints can be of tremendous value for forensic biology, since they can be collected from a wide variety of evident types, such as handles of weapons, tools collected in criminal cases, and objects with no apparent staining. DNA obtained from fingerprints varies greatly in quality and quantity, which ultimately affects the quality of the resulting STR profiles. Additional difficulties can arise when fingerprint samples show mixed STR profiles due to the handling of multiple persons. After applying a tested protocol for sample collection (swabbing with 5% Triton X-100), DNA extraction (using an enzyme that works at elevated temperatures), and PCR amplification (AmpFlSTR® Identifiler® using 31cycles) extensive analysis was performed to better understand the challenges inherent to fingerprint samples, with the ultimate goal of developing valuable profiles (≥50% complete). The impact of time on deposited fingerprints was investigated, revealing that while the quality of profiles deteriorated, full STR profiles could still be obtained from samples after 40days of storage at room temperature. By comparing the STR profiles from fingerprints of the dominant versus the non-dominant hand, we found a slightly better quality from the non-dominant hand, which was not always significant. Substrates seem to have greater effects on fingerprints. Tests on glass, plastic, paper and metal (US Quarter dollar, made of Cu and Ni), common substrates in offices and homes, showed best results for glass, followed by plastic and paper, while almost no profiles were obtained from a Quarter dollar. Important for forensic casework, we also assessed three-person mixtures of touched fingerprint samples. Unlike routinely used approaches for sampling evidence, the surface of an object (bottle) was sectioned into six equal parts and separate samples were taken from each section. The samples were processed separately for DNA extraction and STR amplification. The results included a few single

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

    PubMed Central

    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

    2011-01-01

    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

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

    PubMed Central

    Quirós, Pablo; Colomer-Lluch, Marta; Martínez-Castillo, Alexandre; Miró, Elisenda; Argente, Marc; Jofre, Juan; Navarro, Ferran

    2014-01-01

    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. PMID:24165177

  14. Sample processing and cDNA preparation for microbial metatranscriptomics in complex soil communities.

    PubMed

    Carvalhais, Lilia C; Schenk, Peer M

    2013-01-01

    Soil presents one of the most complex environments for microbial communities as it provides many microhabitats that allow coexistence of thousands of species with important ecosystem functions. These include biomass and nutrient cycling, mineralization, and detoxification. Culture-independent DNA-based methods, such as metagenomics, have revealed operational taxonomic units that suggest a high diversity of microbial species and associated functions in soil. An emerging but technically challenging area to profile the functions of microorganisms and their activities is mRNA-based metatranscriptomics. Here, we describe issues and important considerations of soil sample processing and cDNA preparation for metatranscriptomics from bacteria and archaea and provide a set of methods that can be used in the required experimental steps.

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

    PubMed

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

    2014-01-01

    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 μg 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

  16. Urine stability and steroid profile: towards a screening index of urine sample degradation for anti-doping purpose.

    PubMed

    Mazzarino, Monica; Abate, Maria Gabriella; Alocci, Roberto; Rossi, Francesca; Stinchelli, Raffaella; Molaioni, Francesco; de la Torre, Xavier; Botrè, Francesco

    2011-01-10

    The presence of microorganisms in urine samples, under favourable conditions of storage and transportation, may alter the concentration of steroid hormones, thus altering the correct evaluation of the urinary steroid profile in doping control analysis. According to the rules of the World Anti-Doping Agency (WADA technical document TD2004 EAAS), a testosterone deconjugation higher than 5% and the presence of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione in the deconjugated fraction, are reliable indicators of urine degradation. The determination of these markers would require an additional quantitative analysis since the steroids screening analysis, in anti-doping laboratories, is performed in the total (free+conjugated) fraction. The aim of this work is therefore to establish reliable threshold values for some representative compounds (namely 5α-androstane-3,17-dione and 5β-androstane-3,17-dione) in the total fraction in order to predict directly at the screening stage the potential microbial degradation of the urine samples. Preliminary evidence on the most suitable degradation indexes has been obtained by measuring the urinary concentration of testosterone, epitestosterone, 5α-androstane-3,17-dione and 5β-androstane-3,17-dione by gas chromatography-mass spectrometric every day for 15 days in the deconjugated, glucuronide and total fraction of 10 pools of urines from 60 healthy subjects, stored under different pH and temperature conditions, and isolating the samples with one or more markers of degradation according to the WADA technical document TD2004EAAS. The threshold values for 5α-androstane-3,17-dione and 5β-androstane-3,17-dione were therefore obtained correlating the testosterone deconjugation rate with the urinary concentrations of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione in the total fraction. The threshold values suggested as indexes of urine degradation in the total fraction were: 10 ng mL(-1) for 5α-androstane-3,17-dione

  17. Use of the designation "shedder" in mucosal detection of herpes simplex virus DNA involving repeated sampling

    PubMed Central

    Magaret, Amalia S.; Johnston, Christine; Wald, Anna

    2009-01-01

    Objectives We evaluated two methods to describe detection of HSV from the genital mucosa. Methods We assessed genital swabs from HSV-2 seropositive persons participating in longitudinal studies of HSV DNA detection at the University of Washington Virology Research Clinic. We determined the length of observation period necessary to ensure some HSV detection for most persons. We compared two measures to assess differences in shedding according to HIV status, the shedding rate ratio, defined as the proportion of total samples with detectable HSV in HIV-1 seropositive versus HIV-1 seronegative persons, and the ratio of "shedders", defined as the proportion of persons with any shedding over the interval in HIV-1 seropositive versus HIV-1 seronegative persons. Results While only 17% (51/308) of HSV-2 seropositive persons shed on their first day on study, 77% (238/308) had some genital shedding over 30 days (any HSV DNA detected on genital swabs). Shedding rate ratios for HIV-seropositive versus HIV-seronegative persons varied from SRR=1.42 using 10 samples to SRR=1.35 using 50 samples. The ratio of "shedders" approached 1 as the observation period increased (RS=1.13 using 10 samples to RS=1.01 using 50 samples). In a hypothetical case, the ratio of "shedders" was shown to exceed one when shedding rates were equal. Conclusions Most HSV-2 seropositive persons shed HSV from the genital mucosa. Dichotomization of persons into “shedders” and “nonshedders” or “high” and “low” shedders yields inferences that depend upon sampling interval length. Overall shedding rates provide consistent measures regardless of the number of swabs collected. PMID:19211593

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

    PubMed Central

    Germer, Søren; Holland, Michael J.; Higuchi, Russell

    2000-01-01

    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. PMID:10673283

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

    PubMed Central

    Kuske, Cheryl R.; Banton, Kaysie L.; Adorada, Dante L.; Stark, Peter C.; Hill, Karen K.; Jackson, Paul J.

    1998-01-01

    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

  20. Autosomal and Y-STR analysis of degraded DNA from the 120-year-old skeletal remains of Ezekiel Harper.

    PubMed

    Ambers, Angie; Gill-King, Harrell; Dirkmaat, Dennis; Benjamin, Robert; King, Jonathan; Budowle, Bruce

    2014-03-01

    The 120-year-old skeletal remains of Confederate Civil War soldier Captain Ezekiel "Zeke" Harper were exhumed by court order in January 2011 for DNA analysis. The goal of the DNA testing was to support or refute whether Captain Harper had fathered a son (Earl J. Maxwell) with his Native American maid prior to his murder in 1892. Bones with adequate structural integrity (left tibia, right tibia, right femur, mandible, four teeth) were retrieved from the burial site and sent to the Institute of Applied Genetics in Fort Worth, Texas for analysis. Given the age and condition of the remains, three different extraction methods were used to maximize the probability of DNA recovery. The majority of the DNA isolates from over fifty separate bone sections yielded partial autosomal STR genotypes and partial Y-STR haplotypes. After comparing the partial results for concordance, consensus profiles were generated for comparison to reference samples from alleged family members. Considering the genetic recombination that occurs in autosomal DNA over the generations within a family, Y-STR analysis was determined to be the most appropriate and informative approach for determining potential kinship. Two of Earl J. Maxwell's grandsons submitted buccal samples for comparison. The Y-STR haplotypes obtained from both of these reference samples were identical to each other and to the alleles in Ezekiel Harper's consensus profile at all 17 loci examined. This Y-STR haplotype was not found in either of two major Y-STR population databases (U.S. Y-STR database and YHRD). The fact that the Y-STR haplotype obtained from Ezekiel's skeletal remains and Earl's grandsons is not found in either population database demonstrates its rarity and further supports a paternal lineage relationship among them. Results of the genetic analyses are consistent with the hypothesis that Earl J. Maxwell is the son of Ezekiel Harper.

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

    PubMed

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

    2012-09-01

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

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

    PubMed Central

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

    2010-01-01

    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

  3. Rapid monitoring of sulfur mustard degradation in solution by headspace solid-phase microextraction sampling and gas chromatography mass spectrometry.

    PubMed

    Creek, Jo-Anne M; McAnoy, Andrew M; Brinkworth, Craig S

    2010-12-15

    A method using headspace solid-phase microextraction (HS-SPME) followed by gas chromatography/mass spectrometry (GC/MS) analysis has been developed to gain insight into the degradation of the chemical warfare agent sulfur mustard in solution. Specifically, the described approach simplifies the sample preparation for GC/MS analysis to provide a rapid determination of changes in sulfur mustard abundance. These results were found to be consistent with those obtained using liquid-liquid extraction (LLE) GC/MS. The utility of the described approach was further demonstrated by the investigation of the degradation process in a complex matrix with surfactant added to assist solvation of sulfur mustard. A more rapid reduction in sulfur mustard abundance was observed using the HS-SPME approach with surfactant present and was similar to results from LLE experiments. Significantly, this study demonstrates that HS-SPME can simplify the sample preparation for GC/MS analysis to monitor changes in sulfur mustard abundance in solution more rapidly, and with less solvent and reagent usage than LLE.

  4. Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples

    PubMed Central

    Whale, Alexandra S.; Cowen, Simon; Foy, Carole A.; Huggett, Jim F.

    2013-01-01

    Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA. PMID:23472156

  5. An effective method to purify Plasmodium falciparum DNA directly from clinical blood samples for whole genome high-throughput sequencing.

    PubMed

    Auburn, Sarah; Campino, Susana; Clark, Taane G; Djimde, Abdoulaye A; Zongo, Issaka; Pinches, Robert; Manske, Magnus; Mangano, Valentina; Alcock, Daniel; Anastasi, Elisa; Maslen, Gareth; Macinnis, Bronwyn; Rockett, Kirk; Modiano, David; Newbold, Christopher I; Doumbo, Ogobara K; Ouédraogo, Jean Bosco; Kwiatkowski, Dominic P

    2011-01-01

    Highly parallel sequencing technologies permit cost-effective whole genome sequencing of hundreds of Plasmodium parasites. The ability to sequence clinical Plasmodium samples, extracted directly from patient blood without a culture step, presents a unique opportunity to sample the diversity of "natural" parasite populations in high resolution clinical and epidemiological studies. A major challenge to sequencing clinical Plasmodium samples is the abundance of human DNA, which may substantially reduce the yield of Plasmodium sequence. We tested a range of human white blood cell (WBC) depletion methods on P. falciparum-infected patient samples in search of a method displaying an optimal balance of WBC-removal efficacy, cost, simplicity, and applicability to low resource settings. In the first of a two-part study, combinations of three different WBC depletion methods were tested on 43 patient blood samples in Mali. A two-step combination of Lymphoprep plus Plasmodipur best fitted our requirements, although moderate variability was observed in human DNA quantity. This approach was further assessed in a larger sample of 76 patients from Burkina Faso. WBC-removal efficacy remained high (<30% human DNA in >70% samples) and lower variation was observed in human DNA quantities. In order to assess the Plasmodium sequence yield at different human DNA proportions, 59 samples with up to 60% human DNA contamination were sequenced on the Illumina Genome Analyzer platform. An average ~40-fold coverage of the genome was observed per lane for samples with ≤ 30% human DNA. Even in low resource settings, using a simple two-step combination of Lymphoprep plus Plasmodipur, over 70% of clinical sample preparations should exhibit sufficiently low human DNA quantities to enable ~40-fold sequence coverage of the P. falciparum genome using a single lane on the Illumina Genome Analyzer platform. This approach should greatly facilitate large-scale clinical and epidemiologic studies of P

  6. Long-Term Frozen Storage of Urine Samples: A Trouble to Get PCR Results in Schistosoma spp. DNA Detection?

    PubMed Central

    Fernández-Soto, Pedro; Velasco Tirado, Virginia; Carranza Rodríguez, Cristina; Pérez-Arellano, José Luis; Muro, Antonio

    2013-01-01

    Background Human schistosomiasis remains a serious worldwide public health problem. At present, a sensitive and specific assay for routine diagnosis of schistosome infection is not yet available. The potential for detecting schistosome-derived DNA by PCR-based methods in human clinical samples is currently being investigated as a diagnostic tool with potential application in routine schistosomiasis diagnosis. Collection of diagnostic samples such as stool or blood is usually difficult in some populations. However, urine is a biological sample that can be collected in a non-invasive method, easy to get from people of all ages and easy in management, but as a sample for PCR diagnosis is still not widely used. This could be due to the high variability in the reported efficiency of detection as a result of the high variation in urine samples’ storage or conditions for handling and DNA preservation and extraction methods. Methodology/Principal Findings We evaluate different commercial DNA extraction methods from a series of long-term frozen storage human urine samples from patients with parasitological confirmed schistosomiasis in order to assess the PCR effectiveness for Schistosoma spp. detection. Patientś urine samples were frozen for 18 months up to 7 years until use. Results were compared with those obtained in PCR assays using fresh healthy human urine artificially contaminated with Schistosoma mansoni DNA and urine samples from mice experimentally infected with S. mansoni cercariae stored frozen for at least 12 months before use. PCR results in fresh human artificial urine samples using different DNA based extraction methods were much more effective than those obtained when long-term frozen human urine samples were used as the source of DNA template. Conclusions/Significance Long-term frozen human urine samples are probably not a good source for DNA extraction for use as a template in PCR detection of Schistosoma spp., regardless of the DNA method of

  7. The importance of Guthrie cards and other medical samples for the direct matching of disaster victims using DNA profiling.

    PubMed

    Hartman, D; Benton, L; Morenos, L; Beyer, J; Spiden, M; Stock, A

    2011-02-25

    The identification of disaster victims through the use of DNA analysis is an integral part of any Disaster Victim Identification (DVI) response, regardless of the scale and nature of the disaster. As part of the DVI response to the 2009 Victorian Bushfires Disaster, DNA analysis was performed to assist in the identification of victims through kinship (familial matching to relatives) or direct (self source sample) matching of DNA profiles. Although most of the DNA identifications achieved were to reference samples from relatives, there were a number of DNA identifications (12) made through direct matching. Guthrie cards, which have been collected in Australia over the past 30 years, were used to provide direct reference samples. Of the 236 ante-mortem (AM) samples received, 21 were Guthrie cards and one was a biopsy specimen; all yielding complete DNA profiles when genotyped. This publication describes the use of such Biobanks and medical specimens as a sample source for the recovery of good quality DNA for comparisons to post-mortem (PM) samples.

  8. 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

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

    1991-01-01

    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 PMID:16348574

  9. Comparison between Urine and Cervical Samples for HPV DNA Detection and Typing in Young Women in Colombia.

    PubMed

    Cómbita, Alba Lucía; Gheit, Tarik; González, Paula; Puerto, Devi; Murillo, Raúl Hernando; Montoya, Luisa; Vorsters, Alex; Van Keer, Severien; Van Damme, Pierre; Tommasino, Massimo; Hernández-Suárez, Gustavo; Sánchez, Laura; Herrero, Rolando; Wiesner, Carolina

    2016-09-01

    Urine sampling for HPV DNA detection has been proposed as an effective method for monitoring the impact of HPV vaccination programs; however, conflicting results have been reported. The goal of this study was to evaluate the performance of optimized urine HPV DNA testing in women aged 19 to 25 years. Optimization process included the use of first void urine, immediate mixing of urine with DNA preservative, and the concentration of all HPV DNA, including cell-free DNA fragments. Urine and cervical samples were collected from 535 young women attending cervical screening at health centers from two Colombian cities. HPV DNA detection and genotyping was performed using an HPV type-specific multiplex genotyping assay, which combines multiplex polymerase chain reaction with bead-based Luminex technology. Concordance between HPV DNA detection in urine and cervical samples was determined using kappa statistics and McNemar tests. The accuracy of HPV DNA testing in urine samples was evaluated measuring sensitivity and specificity using as reference the results obtained from cervical samples. Statistical analysis was performed using STATA11.2 software. The findings revealed an overall HPV prevalence of 60.00% in cervical samples and 64.72% in urine samples, HPV-16 being the most frequent HPV type detected in both specimens. Moreover, our results indicate that detection of HPV DNA in first void urine provides similar results to those obtained with cervical samples and can be used to monitor HPV vaccination trials and programs as evidenced by the substantial concordance found for the detection of the four vaccine types. Cancer Prev Res; 9(9); 766-71. ©2016 AACR.

  10. Microcapillary reactors using solid-phase DNA sequencing for direct sample introduction into slab gels.

    PubMed

    Xu, Y; Bruch, R C; Soper, S A

    2000-05-01

    Solid-phase micro-reactors have been prepared in glass capillaries for DNA sequencing applications using slab gel electrophoresis, which consisted of a fused silica capillary (i.d. = 100 microns; o.d. = 365 microns; length = 15 cm; volume = 1.2 microL) that contained a covalently bound biotin molecule. With the addition of streptavidin to the capillary, an anchoring site was produced for the tethering of biotinylated DNA sequencing templates to the wall of the capillary. Using a four-lane, single dye primer chemistry sequencing strategy, the individual tracts were prepared in the capillaries using cycle sequencing (20 thermal cycles) on a PCR-generated lambda-bacteriophage template (about 1000 bp). The dye label in this case was a fluorescent tag that displayed emission properties in the near-IR and could be processed on an automated sequencer. The read length was found to be 589 bases, which was determined primarily by the fractionating power of the gel. It was also found that the tethering system was very stable to typical cycle sequencing conditions, with the amount of tethered DNA lost amounting to 40% after 120 thermal cycles. The ability to use dye terminator chemistry was also investigated by using a near-IR dye-labeled terminator (ddGTP). It was found that the quality of the ladder that was generated was comparable to that obtained in a conventional sample preparation format. However, ethanol precipitation was required before gel loading to remove excess terminator.

  11. Bacteria capable of degrading anthracene, phenanthrene, and fluoranthene as revealed by DNA based stable-isotope probing in a forest soil.

    PubMed

    Song, Mengke; Jiang, Longfei; Zhang, Dayi; Luo, Chunling; Wang, Yan; Yu, Zhiqiang; Yin, Hua; Zhang, Gan

    2016-05-05

    Information on microorganisms possessing the ability to metabolize different polycyclic aromatic hydrocarbons (PAHs) in complex environments helps in understanding PAHs behavior in natural environment and developing bioremediation strategies. In the present study, stable-isotope probing (SIP) was applied to investigate degraders of PAHs in a forest soil with the addition of individually (13)C-labeled phenanthrene, anthracene, and fluoranthene. Three distinct phylotypes were identified as the active phenanthrene-, anthracene- and fluoranthene-degrading bacteria. The putative phenanthrene degraders were classified as belonging to the genus Sphingomona. For anthracene, bacteria of the genus Rhodanobacter were the putative degraders, and in the microcosm amended with fluoranthene, the putative degraders were identified as belonging to the phylum Acidobacteria. Our results from DNA-SIP are the first to directly link Rhodanobacter- and Acidobacteria-related bacteria with anthracene and fluoranthene degradation, respectively. The results also illustrate the specificity and diversity of three- and four-ring PAHs degraders in forest soil, contributes to our understanding on natural PAHs biodegradation processes, and also proves the feasibility and practicality of DNA-based SIP for linking functions with identity especially uncultured microorganisms in complex microbial biota.

  12. NMR studies of bent DNA using {sup 13}C-enriched samples

    SciTech Connect

    Zimmer, D.P.; Crothers, D.M.

    1994-12-01

    Bending of the DNA double helix can be brought about by introducing runs of adenines (A-tracts) in phase with the helical repeat of the DNA. The requirements for bending of DNA by A-tracts are that the length of the A-tract be greater than 3 base pairs and that the A-tracts must be in phase with the helical repeat (every 10 or 11 bp). Other factors, such as the number of adenines in the run, flanking sequences, and whether the A-tracts are phased with respect to the 5{prime}A or the 3{prime}A, have effects upon the degree of bending as assayed by electrophoretic mobility on native polyacrylamide gels. There are a number of models for bending A-tract DNA. The junction-bending model postulates that the structure of A-tracts is similar to the fiber diffraction structure of poly A, in which there is a significant degree of base pair tilt with respect to the helix axis. In this model, bending occurs at the junction between the A-tract and the B-form helix to allow favorable stacking interactions to occur. The bend of the helix could arise as a result of some other perturbation of B-form DNA by A-tracts, such as propeller twist; bending also could be due to a combination of factors. Our goal is to find the structural features of A-tracts responsible for bending of the helix by performing NMR on oligonucleotides containing A-tracts to obtain higher resolution structural data. One of the problems encountered in NMR structure determination of nucleic acids and other macromolecules is the assignment of resonances to nuclei. This procedure can be greatly facilitated through the use of {sup 13}C-enriched nucleic acid samples. We are developing a technique for the enzymatic synthesis of labeled DNA for NMR. The technique we are developing is similar to RNA labeling techniques already in use. The technique involves growth of methylotrophic bacteria on {sup 13}CH{sub 3}OH.

  13. Feline mitochondrial DNA sampling for forensic analysis: When enough is enough!

    PubMed Central

    Grahn, Robert A.; Alhaddad, Hasan; Alves, Paulo C.; Randi, Ettore; Waly, Nashwa E.; Lyons, Leslie A.

    2015-01-01

    Pet hair has a demonstrated value in resolving legal issues. Cat hair is chronically shed and it is difficult to leave a home with cats without some level of secondary transfer. The power of cat hair as an evidentiary resource may be underused because representative genetic databases are not available for exclusionary purposes. Mitochondrial control region databases are highly valuable for hair analyses and have been developed for the cat. In a representative worldwide data set, 83% of domestic cat mitotypes belong to one of twelve major types. Of the remaining 17%, 7.5% are unique within the published 1394 sample database. The current research evaluates the sample size necessary to establish a representative population for forensic comparison of the mitochondrial control region for the domestic cat. For most worldwide populations, randomly sampling 50 unrelated local individuals will achieve saturation at 95%. The 99% saturation is achieved by randomly sampling 60–170 cats, depending on the numbers of mitotypes available in the population at large. Likely due to the recent domestication of the cat and minimal localized population substructure, fewer cats are needed to meet mitochondria DNA control region database practical saturation than for humans or dogs. Coupled with the available worldwide feline control region database of nearly 1400 cats, minimal local sampling will be required to establish an appropriate comparative representative database and achieve significant exclusionary power. PMID:25531059

  14. Feline mitochondrial DNA sampling for forensic analysis: when enough is enough!

    PubMed

    Grahn, Robert A; Alhaddad, Hasan; Alves, Paulo C; Randi, Ettore; Waly, Nashwa E; Lyons, Leslie A

    2015-05-01

    Pet hair has a demonstrated value in resolving legal issues. Cat hair is chronically shed and it is difficult to leave a home with cats without some level of secondary transfer. The power of cat hair as an evidentiary resource may be underused because representative genetic databases are not available for exclusionary purposes. Mitochondrial control region databases are highly valuable for hair analyses and have been developed for the cat. In a representative worldwide data set, 83% of domestic cat mitotypes belong to one of twelve major types. Of the remaining 17%, 7.5% are unique within the published 1394 sample database. The current research evaluates the sample size necessary to establish a representative population for forensic comparison of the mitochondrial control region for the domestic cat. For most worldwide populations, randomly sampling 50 unrelated local individuals will achieve saturation at 95%. The 99% saturation is achieved by randomly sampling 60-170 cats, depending on the numbers of mitotypes available in the population at large. Likely due to the recent domestication of the cat and minimal localized population substructure, fewer cats are needed to meet mitochondria DNA control region database practical saturation than for humans or dogs. Coupled with the available worldwide feline control region database of nearly 1400 cats, minimal local sampling will be required to establish an appropriate comparative representative database and achieve significant exclusionary power.

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

    USGS Publications Warehouse

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

    2013-01-01

    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 (π), 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

  16. Reduced sampling efficiency causes degraded Vernier hyperacuity with normal aging: Vernier acuity in position noise.

    PubMed

    Li, Roger W; Brown, Brian; Edwards, Marion H; Ngo, Charlie V; Chat, Sandy W; Levi, Dennis M

    2012-01-01

    Vernier acuity, a form of visual hyperacuity, is amongst the most precise forms of spatial vision. Under optimal conditions Vernier thresholds are much finer than the inter-photoreceptor distance. Achievement of such high precision is based substantially on cortical computations, most likely in the primary visual cortex. Using stimuli with added positional noise, we show that Vernier processing is reduced with advancing age across a wide range of noise levels. Using an ideal observer model, we are able to characterize the mechanisms underlying age-related loss, and show that the reduction in Vernier acuity can be mainly attributed to the reduction in efficiency of sampling, with no significant change in the level of internal position noise, or spatial distortion, in the visual system.

  17. GC/MS analysis of triclosan and its degradation by-products in wastewater and sludge samples from different treatments.

    PubMed

    Tohidi, Fatemeh; Cai, Zongwei

    2015-08-01

    A gas chromatography/mass spectrometry (GC/MS)-based method was developed for simultaneous determination of triclosan (TCS) and its degradation products including 2,4-dichlorophenol (2,4-DCP), 2,8-dichlorodibenzo-p-dioxin (2,8-DCDD), and methyl triclosan (MTCS) in wastewater and sludge samples. The method provides satisfactory detection limit, accuracy, precision and recovery especially for samples with complicated matrix such as sewage sludge. Liquid-liquid extraction and accelerated solvent extraction (ASE) methods were applied for the extraction, and column chromatography was employed for the sample cleanup. Analysis was performed by GC/MS in the selected ion monitoring (SIM) mode. The method was successfully applied to wastewater and sludge samples from three different municipal wastewater treatment plants (WWTPs). Satisfactory mean recoveries were obtained as 91(±4)-106(±7)%, 82(±3)-87(±4)%, 86(±6)-87(±8)%, and 88(±4)-105(±3)% in wastewater and 88(±5)-96(±8)%, 84(±2)-87(±3)%, 84(±7)-89(±4)%, and 88(±3)-97(±5)% in sludge samples for TCS, 2,4-DCP, 2,8-DCDD, and MTCS, respectively. TCS degradation products were detected based on the type of the wastewater and sludge treatment. 2,8-DCDD was detected in the plant utilizing UV disinfection at the mean level of 20.3(±4.8) ng/L. 2,4-DCP was identified in chemically enhanced primary treatment (CEPT) applying chlorine disinfection at the mean level of 16.8(±4.5) ng/L). Besides, methyl triclosan (MTCS) was detected in the wastewater collected after biological treatment (10.7 ± 3.3 ng/L) as well as in sludge samples that have undergone aerobic digestion at the mean level of 129.3(±17.2) ng/g dry weight (dw).

  18. Improved age determination of blood and teeth samples using a selected set of DNA methylation markers

    PubMed Central

    Kamalandua, Aubeline

    2015-01-01

    Age estimation from DNA methylation markers has seen an exponential growth of interest, not in the least from forensic scientists. The current published assays, however, can still be improved by lowering the number of markers in the assay and by providing more accurate models to predict chronological age. From the published literature we selected 4 age-associated genes (ASPA, PDE4C, ELOVL2, and EDARADD) and determined CpG methylation levels from 206 blood samples of both deceased and living individuals (age range: 0–91 years). This data was subsequently used to compare prediction accuracy with both linear and non-linear regression models. A quadratic regression model in which the methylation levels of ELOVL2 were squared showed the highest accuracy with a Mean Absolute Deviation (MAD) between chronological age and predicted age of 3.75 years and an adjusted R2 of 0.95. No difference in accuracy was observed for samples obtained either from living and deceased individuals or between the 2 genders. In addition, 29 teeth from different individuals (age range: 19–70 years) were analyzed using the same set of markers resulting in a MAD of 4.86 years and an adjusted R2 of 0.74. Cross validation of the results obtained from blood samples demonstrated the robustness and reproducibility of the assay. In conclusion, the set of 4 CpG DNA methylation markers is capable of producing highly accurate age predictions for blood samples from deceased and living individuals PMID:26280308

  19. Design of a protocol for obtaining genomic DNA from saliva using mouthwash: Samples taken from patients with periodontal disease

    PubMed Central

    Mendoza, Ángel Chávez; Volante, Beatriz Buentello; Hernández, María Esther Ocharán; Mendoza, Claudia Camelia Calzada; Pliego, Arturo Flores; Baptista Gonzalez, Héctor A.; Juárez, Higinio Estrada

    2016-01-01

    Background Obtaining high quality genomic DNA safely and economically is vital for diverse studies of large populations aimed at evaluating the role of genetic factors in susceptibility to disease. Aim This study was to test a protocol for the extraction of high quality genomic DNA from saliva samples obtained with mouthwash and taken from patients with periodontal disease. Methods Saliva samples were taken from 60 patients and then stored at room temperature. DNA extraction was carried out at distinct post-sampling times (10, 20 and 30 days). Evaluation of genomic DNA was performed with spectrophotometry, electrophoresis, and PCR genotyping and sequencing. Results The greatest concentration of DNA obtained was 352 μg at 10 days post-sampling, followed by 121.025 μg and 19.59 μg at 20 and 30 days, respectively. When determining the purity of DNA with the spectrophotometric ratio of 260/230, the relations of 1.20, 1.40 and 0.781 were obtained for 10, 20 and 30 days, respectively. In all samples, it was possible to amplify the product of 485 bp and the sequence of the amplicons showed 95% similarity to the reference sequence. Conclusion The present protocol represents an easy, safe and economical technique for obtaining high quality genomic DNA. PMID:27195211

  20. Molecular evidence of Orthopoxvirus DNA in capybara (Hydrochoerus hydrochaeris) stool samples.

    PubMed

    Dutra, Lara Ambrosio Leal; de Freitas Almeida, Gabriel Magno; Oliveira, Graziele Pereira; Abrahão, Jônatas Santos; Kroon, Erna Geessien; Trindade, Giliane de Souza

    2017-02-01

    Vaccinia virus (VACV) is responsible for outbreaks in Brazil and has immense potential as an emerging virus. VACV can be found naturally circulating in India, Pakistan and South America, where it causes infections characterised by exanthematic lesions in buffaloes, cattle and humans. The transmission cycle of Brazilian VACV has still not been fully characterised; one of the most important gaps in knowledge being the role of wild animals. Capybaras, which are restricted to the Americas, are the world's largest rodents and have peculiar characteristics that make them possible candidates for being part of a natural VACV reservoir. Here, we developed a method for detecting orthopoxvirus DNA in capybara stool samples, and have described for the first time the detection of orthopoxvirus DNA in capybaras samples from three different regions in Brazil. These findings strongly suggest that capybaras might be involved in the natural transmission cycle of VACV and furthermore represent a public health problem, when associated with Brazilian bovine vaccinia outbreaks. This makes infected animals an important factor to be considered when predicting and managing Brazilian VACV outbreaks.

  1. Simulated Annealing-Extended Sampling for Multicomponent Decomposition of Spectral Data of DNA Complexed with Peptide

    NASA Astrophysics Data System (ADS)

    Kang, Jiyoung; Yamasaki, Kazuhiko; Sano, Kuniaki; Tsutsui, Ken; Tsutsui, Kimiko M.; Tateno, Masaru

    2017-01-01

    Theoretical analyses of multivariate data have become increasingly important in various scientific disciplines. The multivariate curve resolution alternating least-squares (MCR-ALS) method is an integrated and systematic tool to decompose such various types of spectral data to several pure spectra, corresponding to distinct species. However, in the present study, the MCR-ALS calculation provided only unreasonable solutions, when used to process the circular dichroism spectra of double-stranded DNA (228 bp) in the complex with a DNA-binding peptide under various concentrations. To resolve this problem, we developed an algorithm by including a simulated annealing (SA) protocol (the SA-MCR-ALS method), to facilitate the expansion of the sampling space. The analysis successfully decomposed the aforementioned data into three reasonable pure spectra. Thus, our SA-MCR-ALS scheme provides a useful tool for effective extended sampling, to investigate the substantial and detailed properties of various forms of multivariate data with significant difficulties in the degrees of freedom.

  2. Mechanism of the alkali degradation of (6-4) photoproduct-containing DNA.

    PubMed

    Arichi, Norihito; Inase, Aki; Eto, Sachise; Mizukoshi, Toshimi; Yamamoto, Junpei; Iwai, Shigenori

    2012-03-21

    The (6-4) photoproduct is one of the major damaged bases produced by ultraviolet light in DNA. This lesion is known to be alkali-labile, and strand breaks occur at its sites when UV-irradiated DNA is treated with hot alkali. We have analyzed the product obtained by the alkali treatment of a dinucleoside monophosphate containing the (6-4) photoproduct, by HPLC, NMR spectroscopy, and mass spectrometry. We previously found that the N3-C4 bond of the 5' component was hydrolyzed by a mild alkali treatment, and the present study revealed that the following reaction was the hydrolysis of the glycosidic bond at the 3' component. The sugar moiety of this component was lost, even when a 3'-flanking nucleotide was not present. Glycosidic bond hydrolysis was also observed for a dimer and a trimer containing 5-methyl-2-pyrimidinone, which was used as an analog of the 3' component of the (6-4) photoproduct, and its mechanism was elucidated. Finally, the alkali treatment of a tetramer, d(GT(6-4)TC), yielded 2'-deoxycytidine 5'-monophosphate, while 2'-deoxyguanosine 3'-monophosphate was not detected. This result demonstrated the hydrolysis of the glycosidic bond at the 3' component of the (6-4) photoproduct and the subsequent strand break by β-elimination. It was also shown that the glycosidic bond at the 3' component of the Dewar valence isomer was more alkali-labile than that of the (6-4) photoproduct.

  3. Degraded mitochondrial DNA is a newly identified subtype of the damage associated molecular pattern (DAMP) family and possible trigger of neurodegeneration.

    PubMed

    Mathew, Alex; Lindsley, Tara A; Sheridan, Anna; Bhoiwala, Devang L; Hushmendy, Shazaan F; Yager, Eric J; Ruggiero, Elizabeth A; Crawford, Dana R

    2012-01-01

    We previously showed a preferential degradation and down-regulation of mitochondrial DNA and RNA in hamster fibroblasts in response to hydrogen peroxide. Subsequent studies by others demonstrated that mitochondrial DNA can stimulate immune cells as a DAMP (damage associated molecular patterns) family member. However, the actual physical structure of this mitochondrial DNA DAMP and its importance in non-immune cell types are poorly understood. Here we report that transfected oxidant-initiated degraded mitochondrial polynucleotides, which we term "DeMPs", strongly induce the proinflammatory cytokines interleukin 6, monocyte chemotactic protein-1, and tumor necrosis factor α in mouse primary astrocytes. Additionally, proinflammatory IL1β was induced, implicating DeMPs in inflammasome activation. Furthermore, human cerebrospinal fluid (CSF) and plasma were found to contain detectable DeMP signal. Finally, significant degradation of mitochondrial DNA was observed in response to either a bolus or steady state hydrogen peroxide. Combined, these studies demonstrate, all for the first time, that a pathophysiologically relevant form of mitochondrial DNA (degraded) can elicit a proinflammatory cytokine induction; that a brain cell type (astrocytes) elicits a proinflammatory cytokine induction in response to these DeMPs; that this induction includes the inflammasome; that astrocytes are capable of inflammasome activation by DeMPs; that DeMPs are detectable in CSF and plasma; and that hydrogen peroxide can stimulate an early stage cellular degradation of mitochondrial DNA. These results provide new insights and are supportive of our hypothesis that DeMPs are a newly identified trigger of neurodegenerative diseases such as Alzheimer's disease, which are known to be associated with early stage inflammation and oxidation.

  4. The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics.

    PubMed

    Fattorini, Paolo; Previderè, Carlo; Sorçaburu-Cigliero, Solange; Marrubini, Giorgio; Alù, Milena; Barbaro, Anna M; Carnevali, Eugenia; Carracedo, Angel; Casarino, Lucia; Consoloni, Lara; Corato, Silvia; Domenici, Ranieri; Fabbri, Matteo; Giardina, Emiliano; Grignani, Pierangela; Baldassarra, Stefania Lonero; Moratti, Marco; Nicolin, Vanessa; Pelotti, Susi; Piccinini, Andrea; Pitacco, Paola; Plizza, Laura; Resta, Nicoletta; Ricci, Ugo; Robino, Carlo; Salvaderi, Luca; Scarnicci, Francesca; Schneider, Peter M; Seidita, Gregorio; Trizzino, Lucia; Turchi, Chiara; Turrina, Stefania; Vatta, Paolo; Vecchiotti, Carla; Verzeletti, Andrea; De Stefano, Francesco

    2014-11-01

    genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles.

  5. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples.

    PubMed

    Rothrock, Michael J; Hiett, Kelli L; Gamble, John; Caudill, Andrew C; Cicconi-Hogan, Kellie M; Caporaso, J Gregory

    2014-12-10

    The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the "gold standard" enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

  6. A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

    PubMed Central

    Rothrock, Michael J.; Hiett, Kelli L.; Gamble, John; Caudill, Andrew C.; Cicconi-Hogan, Kellie M.; Caporaso, J. Gregory

    2014-01-01

    The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the “gold standard” enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples. PMID:25548939

  7. Reef-associated crustacean fauna: biodiversity estimates using semi-quantitative sampling and DNA barcoding

    NASA Astrophysics Data System (ADS)

    Plaisance, L.; Knowlton, N.; Paulay, G.; Meyer, C.

    2009-12-01

    The cryptofauna associated with coral reefs accounts for a major part of the biodiversity in these ecosystems but has been largely overlooked in biodiversity estimates because the organisms are hard to collect and identify. We combine a semi-quantitative sampling design and a DNA barcoding approach to provide metrics for the diversity of reef-associated crustacean. Twenty-two similar-sized dead heads of Pocillopora were sampled at 10 m depth from five central Pacific Ocean localities (four atolls in the Northern Line Islands and in Moorea, French Polynesia). All crustaceans were removed, and partial cytochrome oxidase subunit I was sequenced from 403 individuals, yielding 135 distinct taxa using a species-level criterion of 5% similarity. Most crustacean species were rare; 44% of the OTUs were represented by a single individual, and an additional 33% were represented by several specimens found only in one of the five localities. The Northern Line Islands and Moorea shared only 11 OTUs. Total numbers estimated by species richness statistics (Chao1 and ACE) suggest at least 90 species of crustaceans in Moorea and 150 in the Northern Line Islands for this habitat type. However, rarefaction curves for each region failed to approach an asymptote, and Chao1 and ACE estimators did not stabilize after sampling eight heads in Moorea, so even these diversity figures are underestimates. Nevertheless, even this modest sampling effort from a very limited habitat resulted in surprisingly high species numbers.

  8. DNA-wrapped multi-walled carbon nanotube modified electrochemical biosensor for the detection of Escherichia coli from real samples.

    PubMed

    Ozkan-Ariksoysal, Dilsat; Kayran, Yasin Ugur; Yilmaz, Fethiye Ferda; Ciucu, Anton Alexandru; David, Iulia Gabriela; David, Vasile; Hosgor-Limoncu, Mine; Ozsoz, Mehmet

    2017-05-01

    This paper introduces DNA-wrapped multi-walled carbon nanotube (MWCNT)-modified genosensor for the detection of Escherichia coli (E. coli) from polymerase chain reaction (PCR)-amplified real samples while Staphylococcus aureus (S. aureus) was used to investigate the selectivity of the biosensor. The capture probe specifically recognizing E. coli DNA and it was firstly interacted with MWCNTs for wrapping of single-stranded DNA (ssDNA) onto the nanomaterial. DNA-wrapped MWCNTs were then immobilised on the surface of disposable pencil graphite electrode (PGE) for the detection of DNA hybridization. Electrochemical behaviors of the modified PGEs were investigated using Raman spectroscopy and differential pulse voltammetry (DPV). The sequence selective DNA hybridization was determined and evaluated by changes in the intrinsic guanine oxidation signal at about 1.0V by DPV. Numerous factors affecting the hybridization were optimized such as target concentration, hybridization time, etc. The designed DNA sensor can well detect E. coli DNA in 20min detection time with 0.5pmole of detection limit in 30µL of sample volume.

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

    PubMed Central

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

    1993-01-01

    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 PMID:8285690

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

    PubMed

    Bower, Neil I; Johnston, Ian A

    2010-11-01

    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.

  11. Determination of organophosphorus pesticides and their major degradation product residues in food samples by HPLC-UV.

    PubMed

    Peng, Guilong; He, Qiang; Lu, Ying; Mmereki, Daniel; Zhong, Zhihui

    2016-10-01

    A simple method based on dispersive solid-phase extraction (DSPE) and dispersive liquid-liquid microextraction method based on solidification of floating organic droplets (DLLME-SFO) was developed for the extraction of chlorpyrifos (CP), chlorpyrifos-methyl (CPM), and their main degradation product 3,5,6-trichloro-2-pyridinol (TCP) in tomato and cucumber samples. The determination was carried out by high performance liquid chromatography with ultraviolet detection (HPLC-UV). In the DSPE-DLLME-SFO, the analytes were first extracted with acetone. The clean-up of the extract by DSPE was carried out by directly adding activated carbon sorbent into the extract solution, followed by shaking and filtration. Under the optimum conditions, the proposed method was sensitive and showed a good linearity within a range of 2-500 ng/g, with the correlation coefficients (r) varying from 0.9991 to 0.9996. The enrichment factors ranged from 127 to 138. The limit of detections (LODs) were in the range of 0.12-0.68 ng/g, and the relative standard deviations (RSDs) for 50 ng/g of each analytes in tomato samples were in the range of 3.25-6.26 % (n = 5). The proposed method was successfully applied for the extraction and determination of the mentioned analytes residues in tomato and cucumber samples, and satisfactory results were obtained.

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

    PubMed

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

    2013-07-01

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

  13. Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples

    PubMed Central

    Psifidi, Androniki; Dovas, Chrysostomos I.; Bramis, Georgios; Lazou, Thomai; Russel, Claire L.; Arsenos, Georgios; Banos, Georgios

    2015-01-01

    Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals. PMID:25635817

  14. The α2 helix in the DNA ligase IV BRCT-1 domain is required for targeted degradation of ligase IV during adenovirus infection.

    PubMed

    Gilson, Timra; Greer, Amy E; Vindigni, Alessandro; Ketner, Gary; Hanakahi, Leslyn A

    2012-07-05

    In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood. Through genetic analysis, we identified the α2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 α2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 α2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV.

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

    PubMed Central

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

    2011-01-01

    Background A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. Methodology/Findings An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10−3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95

  16. High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique

    SciTech Connect

    Zhang, Yonghua

    2000-01-01

    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

  17. In vitro reconstitution of an Escherichia coli RNA-guided immune system reveals unidirectional, ATP-dependent degradation of DNA target.

    PubMed

    Mulepati, Sabin; Bailey, Scott

    2013-08-02

    Many prokaryotes utilize small RNA transcribed from clustered, regularly interspaced, short palindromic repeats (CRISPRs) to protect themselves from foreign genetic elements, such as phage and plasmids. In Escherichia coli, this small RNA is packaged into a surveillance complex (Cascade) that uses the RNA sequence to direct binding to invasive DNA. Once bound, Cascade recruits the Cas3 nuclease-helicase, which then proceeds to progressively degrade the invading DNA. Here, using individually purified Cascade and Cas3 from E. coli, we reconstitute CRISPR-mediated plasmid degradation in vitro. Analysis of this reconstituted assay suggests that Cascade recruits Cas3 to a single-stranded region of the DNA target exposed by Cascade binding. Cas3 then nicks the exposed DNA. Recruitment and nicking is stimulated by the presence, but not hydrolysis, of ATP. Following nicking and powered by ATP hydrolysis, the concerted actions of the helicase and nuclease domains of Cas3 proceed to unwind and degrade the entire DNA target in a unidirectional manner.

  18. Characterizing the Temporal Dynamics of Human Papillomavirus DNA Detectability Using Short-Interval Sampling

    PubMed Central

    Liu, Su-Hsun; Cummings, Derek AT; Zenilman, Jonathan M; Gravitt, Patti E; Brotman, Rebecca M

    2013-01-01

    Background Variable detection of human papillomavirus (HPV) DNA can result in misclassification of infection status, but the extent of misclassification has not been quantitatively evaluated. Methods In 2005–2007, 33 women aged 22–53 self-collected vaginal swabs twice per week for 16 consecutive weeks. Each of the 955 swabs collected was tested for 37 HPV types/subtypes. Assuming that a woman’s underlying infection status did not change over the short study period, biases in prevalence estimates obtained from single versus multiple swabs were calculated. Using event history analysis methods, time to recurrent gain and loss of at least one HPV type was determined, separately. Baseline any- and high risk-HPV prevalence was 60.6% and 24.2%, respectively. Cumulative any- and high risk-HPV prevalence over the 16-week period was 84.8% and 60.6%, separately. Results Overall, there were 319 events of detection and 313 events of loss of detection. Median times to a recurrent detection and loss of detection was 11 and 7 days, respectively. Neither vaginal sex nor condom use during follow-up was associated with recurrent viral detection or loss of detection. Assuming the cumulative 16-week prevalence reflects the true prevalence of infection, the baseline any-HPV prevalence under-estimated infection status by 24.2%, with a bootstrapped mean of 20.2% (95% confidence interval [CI]: 8.9%, 29.6%). Conclusions These findings suggest that a substantial proportion of HPV-infected women are misclassified as being un-infected when using a single-time DNA measurement. Impact Short-term variation in detectable HPV DNA needs to be considered while interpreting the natural history of infections using single samples collected at long intervals. PMID:24130223

  19. Complexity of genetic sequences modified by horizontal gene transfer and degraded-DNA uptake

    NASA Astrophysics Data System (ADS)

    Tremberger, George; Dehipawala, S.; Nguyen, A.; Cheung, E.; Sullivan, R.; Holden, T.; Lieberman, D.; Cheung, T.

    2015-09-01

    Horizontal gene transfer has been a major vehicle for efficient transfer of genetic materials among living species and could be one of the sources for noncoding DNA incorporation into a genome. Our previous study of lnc- RNA sequence complexity in terms of fractal dimension and information entropy shows a tight regulation among the studied genes in numerous diseases. The role of sequence complexity in horizontal transferred genes was investigated with Mealybug in symbiotic relation with a 139K genome microbe and Deinococcus radiodurans as examples. The fractal dimension and entropy showed correlation R-sq of 0.82 (N = 6) for the studied Deinococcus radiodurans sequences. For comparison the Deinococcus radiodurans oxidative stress tolerant catalase and superoxide dismutase genes under extracellular dGMP growth condition showed R-sq ~ 0.42 (N = 6); and the studied arsenate reductase horizontal transferred genes for toxicity survival in several microorganisms showed no correlation. Simulation results showed that R-sq < 0.4 would be improbable at less than one percent chance, suggestive of additional selection pressure when compared to the R-sq ~ 0.29 (N = 21) in the studied transferred genes in Mealybug. The mild correlation of R-sq ~ 0.5 for fractal dimension versus transcription level in the studied Deinococcus radiodurans sequences upon extracellular dGMP growth condition would suggest that lower fractal dimension with less electron density fluctuation favors higher transcription level.

  20. Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses.

    PubMed

    Lim, Natalie Y N; Roco, Constance A; Frostegård, Åsa

    2016-01-01

    Adequate comparisons of DNA and cDNA libraries from complex environments require methods for co-extraction of DNA and RNA due to the inherent heterogeneity of such samples, or risk bias caused by variations in lysis and extraction efficiencies. Still, there are few methods and kits allowing simultaneous extraction of DNA and RNA from the same sample, and the existing ones generally require optimization. The proprietary nature of kit components, however, makes modifications of individual steps in the manufacturer's recommended procedure difficult. Surprisingly, enzymatic treatments are often performed before purification procedures are complete, which we have identified here as a major problem when seeking efficient genomic DNA removal from RNA extracts. Here, we tested several DNA/RNA co-extraction commercial kits on inhibitor-rich soils, and compared them to a commonly used phenol-chloroform co-extraction method. Since none of the kits/methods co-extracted high-quality nucleic acid material, we optimized the extraction workflow by introducing small but important improvements. In particular, we illustrate the need for extensive purification prior to all enzymatic procedures, with special focus on the DNase digestion step in RNA extraction. These adjustments led to the removal of enzymatic inhibition in RNA extracts and made it possible to reduce genomic DNA to below detectable levels as determined by quantitative PCR. Notably, we confirmed that DNase digestion may not be uniform in replicate extraction reactions, thus the analysis of "representative samples" is insufficient. The modular nature of our workflow protocol allows optimization of individual steps. It also increases focus on additional purification procedures prior to enzymatic processes, in particular DNases, yielding genomic DNA-free RNA extracts suitable for metatranscriptomic analysis.

  1. High-throughput sample-to-answer detection of DNA/RNA in crude samples within functionalized micro-pipette tips.

    PubMed

    Lu, Wenjing; Wang, Jidong; Wu, Qiong; Sun, Jiashu; Chen, Yiping; Zhang, Lu; Zheng, Chunsheng; Gao, Wenna; Liu, Yi; Jiang, Xingyu

    2016-01-15

    We develop a micro-pipette tip-based nucleic acid test (MTNT) for high-throughput sample-to-answer detection of both DNA and RNA from crude samples including cells, bacteria, and solid plants, without the need of sample pretreatment and complex operation. MTNT consists of micro-pipette tips and embedded solid phase nucleic acid extraction membranes, and fully integrates the functions of nucleic acid extraction from crude samples, loop-mediated isothermal amplification (LAMP) of nucleic acids, and visual readout of assays. The total assaying time for DNA or RNA from a variety of crude samples ranges from 90 to 160 min. The limit of detection (LOD) of MTNT is 2 copies of plasmids containing the target nucleic acid fragments of Ebola virus, and 8 CFU of Escherichia coli carrying Ebola virus-derived plasmids. MTNT can also detect CK-19 mRNA from as few as 2 cancer cells without complicated procedures such as RNA extraction and purification. We further demonstrate MTNT in a high-throughput format using an eight-channel pipette and a homemade mini-heater, with a maximum throughput of 40 samples. Compared with other point-of-care (POC) nucleic acid tests (NAT), MTNT could assay both DNA and RNA directly from liquid (cells/bacteria/blood) or solid (plant) samples in a straightforward, sensitive, high-throughput, and containment-free manner, suggesting a considerable promise for low-cost and POC NAT in remote areas.

  2. The impact of different methods of DNA extraction on microbial community measures of BALF samples based on metagenomic data

    PubMed Central

    Wen, Yan; Xiao, Fei; Wang, Chen; Wang, Zhen

    2016-01-01

    Purpose: It is a challenge to find a better microorganisms DNA extraction method for samples taken from the lower airways for metagenomic sequencing, as the concentrations of bacteria in the alveoli and small airways are likely considerably less than that of the mouth or lower digestive tract. Background DNA from the host, and extraction biases can significantly interfere with microbiota assessment and increase the cost of sequencing. This study aimed to develop an optimized DNA extraction method, which would enable a higher concentration of microbial DNA to be extracted from the samples. Methods: We compared the microbiota profiles of the lower airway communities in twelve individuals with IIP. DNA was extracted using three different extraction methods: QIAamp UCP PurePathogen Blood Kit named kit3 in this study, QIAamp UCP Pathogen Mini Kit named kit2, and QIAamp DNA Microbiome Kit named kit1. DNA libraries were constructed according to the manufacturer’s instructions (Illumina). The same workflows from Illumina were used to perform cluster generation, template hybridization, isothermal amplification, linearization, blocking, denaturing, and hybridization of the sequencing primers. Raw data was uploaded to MG-RAST v3 and analyzed. Results: A great number of bacterium inhabits the lower airways of patients with IIP, though there is no airway infection. More bacterium was found in mouth or upper airway. DNA concentrations of DNA samples isolated with kit1 with Benzonase were significantly lower than those isolated with the other two kits for BALF and mouthwash samples. Moreover, the ratio of human genome in clean reads of samples isolated with kit1 with Benzonase was remarkably smaller than those isolated with kit2 and kit3. The relative abundance of total bacteria, the total number of taxa, and the relative abundance of taxa in BALF samples as opposed to mouthwash samples with kit1 were significantly higher than for those extracted the other kits. Conclusion: A

  3. Comparative study of seven commercial kits for human DNA extraction from urine samples suitable for DNA biomarker-based public health studies.

    PubMed

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

    2014-12-01

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

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

    PubMed Central

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

    2014-01-01

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

  5. Simultaneous detection of BRCA mutations and large genomic rearrangements in germline DNA and FFPE tumor samples

    PubMed Central

    Enyedi, Márton Zsolt; Jaksa, Gábor; Pintér, Lajos; Sükösd, Farkas; Gyuris, Zoltán; Hajdu, Adrienn; Határvölgyi, Erika; Priskin, Katalin; Haracska, Lajos

    2016-01-01

    The development of breast and ovarian cancer is strongly connected to the inactivation of the BRCA1 and BRCA2 genes by different germline and somatic alterations, and their diagnosis has great significance in targeted tumor therapy, since recently approved PARP inhibitors show high efficiency in the treatment of BRCA-deficient tumors. This raises the need for new diagnostic methods that are capable of performing an integrative mutation analysis of the BRCA genes not only from germline DNA but also from formalin-fixed and paraffin-embedded (FFPE) tumor samples. Here we describe the development of such a methodology based on next-generation sequencing and a new bioinformatics software for data analysis. The diagnostic method was initially developed on an Illumina MiSeq NGS platform using germline-mutated stem cell lines and then adapted for the Ion Torrent PGM NGS platform as well. We also investigated the usability of NGS coverage data for the detection of copy number variations and exon deletions as a replacement of the conventional MLPA technique. Finally, we tested the developed workflow on FFPE samples from breast and ovarian cancer patients. Our method meets the sensitivity and specificity requirements for the genetic diagnosis of breast and ovarian cancers both from germline and FFPE samples. PMID:27533253

  6. Developmental toxicity and DNA damage from exposure to parking lot runoff retention pond samples in the Japanese medaka (Oryzias latipes).

    PubMed

    Colton, Meryl D; Kwok, Kevin W H; Brandon, Jennifer A; Warren, Isaac H; Ryde, Ian T; Cooper, Ellen M; Hinton, David E; Rittschof, Daniel; Meyer, Joel N

    2014-08-01

    Parking lot runoff retention ponds (PLRRP) receive significant chemical input, but the biological effects of parking lot runoff are not well understood. We used the Japanese medaka (Oryzias latipes) as a model to study the toxicity of water and sediment samples from a PLRRP in Morehead City, NC. Medaka exposed in ovo to a dilution series of PLRRP water had increased odds of death before hatching, but not teratogenesis or delayed hatching. Next, we adapted a long-amplicon quantitative PCR (LA-QPCR) assay for DNA damage for use with the Japanese medaka. We employed LA-QPCR to test the hypotheses that PLRRP water and sediments would cause nuclear and mitochondrial DNA damage with and without full-spectrum, natural solar radiation. Fluoranthene with and without natural sunlight was a positive control for phototoxic polycyclic aromatic hydrocarbon-induced DNA damage. Fluoranthene exposure did not result in detectable DNA damage by itself, but in combination with sunlight caused significant DNA damage to both genomes. PLRRP samples caused DNA damage to both genomes, and this was not increased by sunlight exposure, suggesting the DNA damage was unlikely the result of PAH phototoxicity. We report for the first time that PLRRP-associated pollutants cause both nuclear and mitochondrial DNA damage, and that fluoranthene-mediated phototoxicity results in similar levels of damage to the nuclear and mitochondrial genomes. These effects may be especially significant in sensitive marine ecosystems.

  7. An automated sample preparation system with mini-reactor to isolate and process submegabase fragments of bacterial DNA.

    PubMed

    Mollova, Emilia T; Patil, Vishal A; Protozanova, Ekaterina; Zhang, Meng; Gilmanshin, Rudolf

    2009-08-15

    Existing methods for extraction and processing of large fragments of bacterial genomic DNA are manual, time-consuming, and prone to variability in DNA quality and recovery. To solve these problems, we have designed and built an automated fluidic system with a mini-reactor. Balancing flows through and tangential to the ultrafiltration membrane in the reactor, cells and then released DNA can be immobilized and subjected to a series of consecutive processing steps. The steps may include enzymatic reactions, tag hybridization, buffer exchange, and selective removal of cell debris and by-products of the reactions. The system can produce long DNA fragments (up to 0.5 Mb) of bacterial genome restriction digest and perform DNA tagging with fluorescent sequence-specific probes. The DNA obtained is of high purity and floating free in solution, and it can be directly analyzed by pulsed-field gel electrophoresis (PFGE) or used in applications requiring submegabase DNA fragments. PFGE-ready samples of DNA restriction digests can be produced in as little as 2.1 h and require less than 10(8) cells. All fluidic operations are automated except for the injection of the sample and reagents.

  8. Developmental toxicity and DNA damage from exposure to parking lot runoff retention pond samples in the Japanese Medaka (Oryzias latipes)

    PubMed Central

    Colton, Meryl D.; Kwok, Kevin W.H.; Brandon, Jennifer A.; Warren, Isaac H.; Ryde, Ian T.; Cooper, Ellen M.; Hinton, David E.; Rittschof, Daniel; Meyer, Joel N.

    2015-01-01

    Parking lot runoff retention ponds (PLRRP) receive significant chemical input, but the biological effects of parking lot runoff are not well understood. We used the Japanese medaka (Oryzias latipes) as a model to study the toxicity of water and sediment samples from a PLRRP in Morehead City, NC. Medaka exposed in ovo to a dilution series of PLRRP water had increased odds of death before hatching, but not teratogenesis or delayed hatching. Next, we adapted a long-amplicon quantitative PCR (LA-QPCR) assay for DNA damage for use with the Japanese medaka. We employed LA-QPCR to test the hypotheses that PLRRP water and sediments would cause nuclear and mitochondrial DNA damage with and without full-spectrum, natural solar radiation. Fluoranthene with and without natural sunlight was a positive control for phototoxic polycyclic aromatic hydrocarbon-induced DNA damage. Fluoranthene exposure did not result in detectable DNA damage by itself, but in combination with sunlight caused significant DNA damage to both genomes. PLRRP samples caused DNA damage to both genomes, and this was not increased by sunlight exposure, suggesting the DNA damage was unlikely the result of PAH phototoxicity. We report for the first time that PLRRP-associated pollutants cause both nuclear and mitochondrial DNA damage, and that fluoranthene-mediated phototoxicity results in similar levels of damage to the nuclear and mitochondrial genomes. These effects may be especially significant in sensitive marine ecosystems. PMID:24816191

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

    PubMed

    Psifidi, Androniki; Dovas, Chrysostomos I; Banos, Georgios

    2010-04-01

    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 1 and 2 were direct applications of two commercial kits, Nucleospin((R)) Blood and Nucleospin((R)) Tissue, respectively. Methods 3 and 4 were based on modified protocols of methods 1 and 2, respectively, aiming at increasing DNA recovery and integrity, and eliminating PCR inhibitors. Method 5 was a standard Phenol-Chloroform protocol application and method 6 was based on an in-house developed protocol using silica as the affinity matrix. Spectrophotometer, gel electrophoresis and real-time PCR measurements were used as criteria for evaluating quantity and quality of the extracted DNA. Processing time, intensity of labor and cost for each method were also evaluated. Results suggested that methods 1-4 were considered suitable for molecular downstream applications and performed better than methods 5 and 6. Modifications of protocols 3 and 4 increased the quantity and quality of the extracted DNA from ovine milk samples. Method 3 was proved to be highly efficient and robust for large scale use as demonstrated by its successful application to 1000 individual ovine milk and 50 bulk milk samples.

  10. High Resolution DNA Stable Isotope Probing Reveals that Root Exudate Addition to Soil Changes the Identity of the Microbes that Degrade Cellulose but not the Rate of Degradation

    NASA Astrophysics Data System (ADS)

    Campbell, A.; Pepe-Ranney, C. P.; Nguyen, A. V. T.; Buckley, D. H.

    2015-12-01

    Plant roots release compounds, such as root exudates, which can alter soil organic matter (SOM) decomposition and have large impacts on soil carbon (C) retention. The changes in SOM turnover resulting from the addition of organic and/or inorganic substrates are termed 'priming effects'. In this study we examine the effects of root exudates on the priming of cellulose added as particulate organic matter. We amended soil microcosms with 13C-cellulose in the presence or absence of artificial root exudate additions and incubated over time for 45 days. Soils receiving the root exudate (RE) were given either one large dose or multiple, small doses of RE. In each treatment we tracked operational taxonomic units (OTUs) assimilating 13C from cellulose (herein, known as a 'responder') over time using DNA stable isotope probing coupled with next generation sequencing. In all treatments the same amount of cellulose-13C was respired indicating the addition of RE did not result in the priming of cellulose decomposition. However, cellulose responders were different depending on treatment and time of sampling (days 14, 28 and 45). We identified a total of 10,361 OTUs, of which there were 369 cellulose responders in the cellulose only treatment, 273 in the repeated, small dose RE treatment, and 358 in the RE single, large dose treatment. Most of the cellulose responders found in all treatments belonged to phyla Bacteroidetes, Planctomycetes, Proteobacteria, Verrucomicrobia, and Chloroflexi. The response time of phyla varies; for instance, more OTUs in Bacteroidetes were observed on day 14 and diminish with each subsequent sampling time. On the other hand, OTUs in Verrucomicrobia increased in response over time. Our study shows no priming effect resulting from the addition of root exudates, although the identity of the microbial mediators of cellulose decomposition varies in each treatment.

  11. Effects of 16S rDNA sampling on estimates of the number of endosymbiont lineages in sucking lice

    PubMed Central

    Burleigh, J. Gordon; Light, Jessica E.; Reed, David L.

    2016-01-01

    Phylogenetic trees can reveal the origins of endosymbiotic lineages of bacteria and detect patterns of co-evolution with their hosts. Although taxon sampling can greatly affect phylogenetic and co-evolutionary inference, most hypotheses of endosymbiont relationships are based on few available bacterial sequences. Here we examined how different sampling strategies of Gammaproteobacteria sequences affect estimates of the number of endosymbiont lineages in parasitic sucking lice (Insecta: Phthirapatera: Anoplura). We estimated the number of louse endosymbiont lineages using both newly obtained and previously sequenced 16S rDNA bacterial sequences and more than 42,000 16S rDNA sequences from other Gammaproteobacteria. We also performed parametric and nonparametric bootstrapping experiments to examine the effects of phylogenetic error and uncertainty on these estimates. Sampling of 16S rDNA sequences affects the estimates of endosymbiont diversity in sucking lice until we reach a threshold of genetic diversity, the size of which depends on the sampling strategy. Sampling by maximizing the diversity of 16S rDNA sequences is more efficient than randomly sampling available 16S rDNA sequences. Although simulation results validate estimates of multiple endosymbiont lineages in sucking lice, the bootstrap results suggest that the precise number of endosymbiont origins is still uncertain. PMID:27547523

  12. Development of a Novel Self-Enclosed Sample Preparation Device for DNA/RNA Isolation in Space

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Mehta, Satish K.; Pensinger, Stuart J.; Pickering, Karen D.

    2011-01-01

    Modern biology techniques present potentials for a wide range of molecular, cellular, and biochemistry applications in space, including detection of infectious pathogens and environmental contaminations, monitoring of drug-resistant microbial and dangerous mutations, identification of new phenotypes of microbial and new life species. However, one of the major technological blockades in enabling these technologies in space is a lack of devices for sample preparation in the space environment. To overcome such an obstacle, we constructed a prototype of a DNA/RNA isolation device based on our novel designs documented in the NASA New Technology Reporting System (MSC-24811-1/3-1). This device is self-enclosed and pipette free, purposely designed for use in the absence of gravity. Our design can also be modified easily for preparing samples in space for other applications, such as flowcytometry, immunostaining, cell separation, sample purification and separation according to its size and charges, sample chemical labeling, and sample purification. The prototype of our DNA/RNA isolation device was tested for efficiencies of DNA and RNA isolation from various cell types for PCR analysis. The purity and integrity of purified DNA and RNA were determined as well. Results showed that our developed DNA/RNA isolation device offers similar efficiency and quality in comparison to the samples prepared using the standard protocol in the laboratory.

  13. Transcriptional profiling of breast cancer cells in response to mevinolin: Evidence of cell cycle arrest, DNA degradation and apoptosis

    PubMed Central

    MAHMOUD, ALI M.; ABOUL-SOUD, MOURAD A.M.; HAN, JUNKYU; AL-SHEIKH, YAZEED A.; AL-ABD, AHMED M.; EL-SHEMY, HANY A.

    2016-01-01

    The merging of high-throughput gene expression techniques, such as microarray, in the screening of natural products as anticancer agents, is considered the optimal solution for gaining a better understanding of the intervention mechanism. Red yeast rice (RYR), a Chinese dietary product, contains a mixture of hypocholesterolemia agents such as statins. Typically, statins have this effect via the inhibition of HMG-CoA reductase, the key enzyme in the biosynthesis of cholesterol. Recently, statins have been shown to exhibit various beneficial antineoplastic properties through the disruption of tumor angiogenesis and metastatic processes. Mevinolin (MVN) is a member of statins and is abundantly present in RYR. Early experimental trials suggested that the mixed apoptotic/necrotic cell death pathway is activated in response to MVN exposure. In the current study, the cytotoxic profile of MVN was evaluated against MCF-7, a breast cancer-derived cell line. The obtained results indicated that MVN-induced cytotoxicity is multi-factorial involving several regulatory pathways in the cytotoxic effects of MVN on breast cancer cell lines. In addition, MVN-induced transcript abundance profiles inferred from microarrays showed significant changes in some key cell processes. The changes were predicted to induce cell cycle arrest and reactive oxygen species generation but inhibit DNA repair and cell proliferation. This MVN-mediated multi-factorial stress triggered specific programmed cell death (apoptosis) and DNA degradation responses in breast cancer cells. Taken together, the observed MVN-induced effects underscore the potential of this ubiquitous natural compound as a selective anticancer activity, with broad safety margins and low cost compared to benchmarked traditional synthetic chemotherapeutic agents. Additionally, the data support further pre-clinical and clinical evaluations of MVN as a novel strategy to combat breast cancer and overcome drug resistance. PMID:26983896

  14. Transcriptional profiling of breast cancer cells in response to mevinolin: Evidence of cell cycle arrest, DNA degradation and apoptosis.

    PubMed

    Mahmoud, Ali M; Aboul-Soud, Mourad A M; Han, Junkyu; Al-Sheikh, Yazeed A; Al-Abd, Ahmed M; El-Shemy, Hany A

    2016-05-01

    The merging of high-throughput gene expression techniques, such as microarray, in the screening of natural products as anticancer agents, is considered the optimal solution for gaining a better understanding of the intervention mechanism. Red yeast rice (RYR), a Chinese dietary product, contains a mixture of hypocholesterolemia agents such as statins. Typically, statins have this effect via the inhibition of HMG‑CoA reductase, the key enzyme in the biosynthesis of cholesterol. Recently, statins have been shown to exhibit various beneficial antineoplastic properties through the disruption of tumor angiogenesis and metastatic processes. Mevinolin (MVN) is a member of statins and is abundantly present in RYR. Early experimental trials suggested that the mixed apoptotic/necrotic cell death pathway is activated in response to MVN exposure. In the current study, the cytotoxic profile of MVN was evaluated against MCF‑7, a breast cancer‑derived cell line. The obtained results indicated that MVN‑induced cytotoxicity is multi‑factorial involving several regulatory pathways in the cytotoxic effects of MVN on breast cancer cell lines. In addition, MVN‑induced transcript abundance profiles inferred from microarrays showed significant changes in some key cell processes. The changes were predicted to induce cell cycle arrest and reactive oxygen species generation but inhibit DNA repair and cell proliferation. This MVN‑mediated multi‑factorial stress triggered specific programmed cell death (apoptosis) and DNA degradation responses in breast cancer cells. Taken together, the observed MVN‑induced effects underscore the potential of this ubiquitous natural compound as a selective anticancer activity, with broad safety margins and low cost compared to benchmarked traditional synthetic chemotherapeutic agents. Additionally, the data support further pre‑clinical and clinical evaluations of MVN as a novel strategy to combat breast cancer and overcome drug resistance.

  15. Inferring separate parental admixture components in unknown DNA samples using autosomal SNPs.

    PubMed

    Crouch, Daniel J M; Weale, Michael E

    2012-12-01

    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.

  16. Transparent DNA/RNA Co-extraction Workflow Protocol Suitable for Inhibitor-Rich Environmental Samples That Focuses on Complete DNA Removal for Transcriptomic Analyses

    PubMed Central

    Lim, Natalie Y. N.; Roco, Constance A.; Frostegård, Åsa

    2016-01-01

    Adequate comparisons of DNA and cDNA libraries from complex environments require methods for co-extraction of DNA and RNA due to the inherent heterogeneity of such samples, or risk bias caused by variations in lysis and extraction efficiencies. Still, there are few methods and kits allowing simultaneous extraction of DNA and RNA from the same sample, and the existing ones generally require optimization. The proprietary nature of kit components, however, makes modifications of individual steps in the manufacturer’s recommended procedure difficult. Surprisingly, enzymatic treatments are often performed before purification procedures are complete, which we have identified here as a major problem when seeking efficient genomic DNA removal from RNA extracts. Here, we tested several DNA/RNA co-extraction commercial kits on inhibitor-rich soils, and compared them to a commonly used phenol-chloroform co-extraction method. Since none of the kits/methods co-extracted high-quality nucleic acid material, we optimized the extraction workflow by introducing small but important improvements. In particular, we illustrate the need for extensive purification prior to all enzymatic procedures, with special focus on the DNase digestion step in RNA extraction. These adjustments led to the removal of enzymatic inhibition in RNA extracts and made it possible to reduce genomic DNA to below detectable levels as determined by quantitative PCR. Notably, we confirmed that DNase digestion may not be uniform in replicate extraction reactions, thus the analysis of “representative samples” is insufficient. The modular nature of our workflow protocol allows optimization of individual steps. It also increases focus on additional purification procedures prior to enzymatic processes, in particular DNases, yielding genomic DNA-free RNA extracts suitable for metatranscriptomic analysis. PMID:27803690

  17. Human DNA Ligase I Interacts with and Is Targeted for Degradation by the DCAF7 Specificity Factor of the Cul4-DDB1 Ubiquitin Ligase Complex.

    PubMed

    Peng, Zhimin; Liao, Zhongping; Matsumoto, Yoshihiro; Yang, Austin; Tomkinson, Alan E

    2016-10-14

    The synthesis, processing, and joining of Okazaki fragments during DNA replication is complex, requiring the sequential action of a large number of proteins. Proliferating cell nuclear antigen, a DNA sliding clamp, interacts with and coordinates the activity of several DNA replication proteins, including the enzymes flap endonuclease 1 (FEN-1) and DNA ligase I that complete the processing and joining of Okazaki fragments, respectively. Although it is evident that maintaining the appropriate relative stoichiometry of FEN-1 and DNA ligase I, which compete for binding to proliferating cell nuclear antigen, is critical to prevent genomic instability, little is known about how the steady state levels of DNA replication proteins are regulated, in particular the proteolytic mechanisms involved in their turnover. Because DNA ligase I has been reported to be ubiquitylated, we used a proteomic approach to map ubiquitylation sites and screen for DNA ligase I-associated E3 ubiquitin ligases. We identified three ubiquitylated lysine residues and showed that DNA ligase I interacts with and is targeted for ubiquitylation by DCAF7, a specificity factor for the Cul4-DDB1 complex. Notably, knockdown of DCAF7 reduced the degradation of DNA ligase I in response to inhibition of proliferation and replacement of ubiquitylated lysine residues reduced the in vitro ubiquitylation of DNA ligase I by Cul4-DDB1 and DCAF7. In contrast, a different E3 ubiquitin ligase regulates FEN-1 turnover. Thus, although the expression of many of the genes encoding DNA replication proteins is coordinately regulated, our studies reveal that different mechanisms are involved in the turnover of these proteins.

  18. CRL4Cdt2 E3 ubiquitin ligase and proliferating cell nuclear antigen (PCNA) cooperate to degrade thymine DNA glycosylase in S phase.

    PubMed

    Shibata, Etsuko; Dar, Ashraf; Dutta, Anindya

    2014-08-15

    Thymine DNA glycosylase (TDG) is an essential enzyme playing multiple roles in base excision repair, transcription regulation, and DNA demethylation. TDG mediates the cytotoxicity of the anti-cancer chemotherapeutic drug 5-fluorouracil (5-FU) by prolonging S phase, generating DNA strand breaks, and inducing DNA damage signaling. During S phase of the cell cycle, TDG is degraded via the proteasomal pathway. Here we show that CRL4(Cdt2) E3 ubiquitin ligase promotes ubiquitination and proteasomal degradation of TDG in S phase in a reaction that is dependent on the interaction of TDG with proliferating cell nuclear antigen (PCNA). siRNA-mediated depletion of PCNA or components of CRL4(Cdt2), specifically cullin4A/B or substrate adaptor Cdt2, stabilizes TDG in human cells. Mutations in the PCNA-interacting peptide (PIP) motif of TDG that disrupt the interaction of TDG with PCNA or change critical basic residues essential for the action of the PIP degron prevent the ubiquitination and degradation of TDG. Thus physical interaction of TDG with PCNA through the PIP degron is required for targeting TDG to the CRL4(Cdt2) E3 ubiquitin ligase complex. Compared with forced expression of wild type TDG, CRL4(Cdt2)- resistant TDG (ΔPIP) slows cell proliferation and slightly increases the toxicity of 5-FU. Thus, CRL4(Cdt2)-dependent degradation of TDG occurs in S phase because of the requirement for TDG to interact with chromatin-loaded PCNA, and this degradation is important for preventing toxicity from excess TDG.

  19. Hepatitis B virus DNA stability in plasma samples under short-term storage at 42°C

    PubMed Central

    de Almeida, R.W.; Espírito-Santo, M.P.; Sousa, P.S.F.; de Almeida, A.J.; Lampe, E.; Lewis-Ximenez, L.L.

    2015-01-01

    We evaluated the stability of hepatitis B virus (HBV) DNA in plasma samples stored at 42°C for external quality assessment (EQA) panels of viral load. To assess the stability of plasma samples containing different concentrations of HBV DNA, serial dilutions of HBV-infected samples with a viral load of 6.40 log(10) IU/mL were made to yield viral loads of 5, 4, and 3 log(10) IU/mL. These were incubated at 42°C for up to 7 days and then frozen at -70°C. Viral load testing for HBV DNA was performed for all samples using COBAS¯ AmpliPrep/COBAS¯ TaqMan¯ HBV Test (v.2.0, Roche, Switzerland). Results were compared with fresh frozen plasma samples as a benchmark to establish acceptable measurements on the days following sample collection. Although the results of this study demonstrated a decrease in HBV DNA viral load ranging from 0.005 to 0.30 log(10) IU/mL after storage at 42°C for up to 7 days, these values did not exceed 0.5 log(10), which is the estimated intra-assay variation for molecular tests. Thus, the insignificant decrease in viral load suggests that shipment of HBV in plasma samples at temperatures of up to 42°C is permissible if they are frozen within 7 days. PMID:25790101

  20. Species Identification in Malaise Trap Samples by DNA Barcoding Based on NGS Technologies and a Scoring Matrix

    PubMed Central

    Morinière, Jérôme; Cancian de Araujo, Bruno; Hausmann, Axel; Balke, Michael; Hendrich, Lars; Doczkal, Dieter; Arvidsson, Samuel; Haszprunar, Gerhard

    2016-01-01

    The German Barcoding initiatives BFB and GBOL have generated a reference library of more than 16,000 metazoan species, which is now ready for applications concerning next generation molecular biodiversity assessments. To streamline the barcoding process, we have developed a meta-barcoding pipeline: We pre-sorted a single malaise trap sample (obtained during one week in August 2014, southern Germany) into 12 arthropod orders and extracted DNA from pooled individuals of each order separately, in order to facilitate DNA extraction and avoid time consuming single specimen selection. Aliquots of each ordinal-level DNA extract were combined to roughly simulate a DNA extract from a non-sorted malaise sample. Each DNA extract was amplified using four primer sets targeting the CO1-5’ fragment. The resulting PCR products (150-400bp) were sequenced separately on an Illumina Mi-SEQ platform, resulting in 1.5 million sequences and 5,500 clusters (coverage ≥10; CD-HIT-EST, 98%). Using a total of 120,000 DNA barcodes of identified, Central European Hymenoptera, Coleoptera, Diptera, and Lepidoptera downloaded from BOLD we established a reference sequence database for a local CUSTOM BLAST. This allowed us to identify 529 Barcode Index Numbers (BINs) from our sequence clusters derived from pooled Malaise trap samples. We introduce a scoring matrix based on the sequence match percentages of each amplicon in order to gain plausibility for each detected BIN, leading to 390 high score BINs in the sorted samples; whereas 268 of these high score BINs (69%) could be identified in the combined sample. The results indicate that a time consuming presorting process will yield approximately 30% more high score BINs compared to the non-sorted sample in our case. These promising results indicate that a fast, efficient and reliable analysis of next generation data from malaise trap samples can be achieved using this pipeline. PMID:27191722

  1. Partition enrichment of nucleotide sequences (PINS)--a generally applicable, sequence based method for enrichment of complex DNA samples.

    PubMed

    Kvist, Thomas; Sondt-Marcussen, Line; Mikkelsen, Marie Just

    2014-01-01

    The dwindling cost of DNA sequencing is driving transformative changes in various biological disciplines including medicine, thus resulting in an increased need for routine sequencing. Preparation of samples suitable for sequencing is the starting point of any practical application, but enrichment of the target sequence over background DNA is often laborious and of limited sensitivity thereby limiting the usefulness of sequencing. The present paper describes a new method, Probability directed Isolation of Nucleic acid Sequences (PINS), for enrichment of DNA, enabling the sequencing of a large DNA region surrounding a small known sequence. A 275,000 fold enrichment of a target DNA sample containing integrated human papilloma virus is demonstrated. Specifically, a sample containing 0.0028 copies of target sequence per ng of total DNA was enriched to 786 copies per ng. The starting concentration of 0.0028 target copies per ng corresponds to one copy of target in a background of 100,000 complete human genomes. The enriched sample was subsequently amplified using rapid genome walking and the resulting DNA sequence revealed not only the sequence of a the truncated virus, but also 1026 base pairs 5' and 50 base pairs 3' to the integration site in chromosome 8. The demonstrated enrichment method is extremely sensitive and selective and requires only minimal knowledge of the sequence to be enriched and will therefore enable sequencing where the target concentration relative to background is too low to allow the use of other sample preparation methods or where significant parts of the target sequence is unknown.

  2. New procedure for recovering extra- and intracellular DNA from marine sediment samples

    NASA Astrophysics Data System (ADS)

    Alawi, M.; Kallmeyer, J.

    2012-12-01

    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. <1μg 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

  3. Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing

    PubMed Central

    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

    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. PMID:24039948

  4. New type of SSUrDNA sequence was detected from both Plasmodium ovale curtisi and Plasmodium ovale wallikeri samples

    PubMed Central

    2014-01-01

    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

  5. Previous estimates of mitochondrial DNA mutation level variance did not account for sampling error: comparing the mtDNA genetic bottleneck in mice and humans.

    PubMed

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

    2010-04-09

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

  6. Effects of ascorbic acid on sperm motility, viability, acrosome reaction and DNA integrity in teratozoospermic samples

    PubMed Central

    Fanaei, Hamed; Khayat, Samira; Halvaei, Iman; Ramezani, Vahid; Azizi, Yaser; Kasaeian, Amir; Mardaneh, Jalal; Parvizi, Mohammad Reza; Akrami, Maryam

    2014-01-01

    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. PMID

  7. Legionella pneumophila DNA in serum samples during Legionnaires' disease in relation to C-reactive protein levels.

    PubMed

    van de Veerdonk, F L; de Jager, C P C; Schellekens, J J A; Huijsmans, C J J; Beaumont, F; Hermans, M H A; Wever, P C

    2009-04-01

    Legionella pneumophila DNA can be detected in serum from patients with Legionnaires' disease (LD). We explored this observation studying the kinetics of L. pneumophila DNA in serum samples in relation to C-reactive protein (CRP). Eleven hospitalized patients with LD were studied. Diagnosis was made by Legionella urinary antigen test in 8 patients and seroconversion in 3 patients. A macrophage infectivity potentiator (MIP) real-time PCR was performed on 31 serum samples, including 20 follow-up serum samples. Serum samples obtained on the day of admission were MIP PCR-positive in 7 (64%) and MIP PCR-negative in 4 (36%) patients. Three (75%) of the 4 patients with a MIP PCR-negative serum sample on the day of admission became positive during follow-up. Overall, L. pneumophila DNA was detected in serum samples from 10 of the 11 patients (91%). CRP levels in the 7 patients with a positive MIP PCR serum sample on day of admission (499 +/- 144 mg/l; median +/- SD) were significantly higher than those in the 4 patients with a negative MIP PCR serum sample on the day of admission (244 +/- 97 mg/l). No difference in the severity of the disease on the day of admission was found between these patients. The presence of L. pneumophila DNA in serum is a common phenomenon in hospitalized patients with LD, although in some cases it is not yet present on the day of admission. L. pneumophila DNA in serum on the day of admission correlates with high CRP levels, but not with the severity of the disease.

  8. Evaluation of DNA extraction techniques for detecting Mycobacterium tuberculosis complex organisms in Asian elephant trunk wash samples.

    PubMed

    Kay, Meagan K; Linke, Lyndsey; Triantis, Joni; Salman, M D; Larsen, R Scott

    2011-02-01

    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.

  9. Rapid Changes in Circulating Tumor DNA in Serially Sampled Plasma During Treatment of Breast Cancer: A Case Report

    PubMed Central

    Nakagomi, Hiroshi; Hirotsu, Yosuke; Amemiya, Kenji; Nakada, Haruka; Inoue, Masayuki; Mochizuki, Hitoshi; Oyama, Toshio; Omata, Masao

    2017-01-01

    Patient: Female, 45 Final Diagnosis: Breast cancer Symptoms: Breast tumor Medication: — Clinical Procedure: Analysis of circulating tumor DNA Specialty: Oncology Objective: Unusual setting of medical care Background: The analysis of circulating tumor DNA (ctDNA) is expected to be a modality to determine the status of cancer in real time. This case indicated utilities and issues in measuring the ctDNA in cancer patients. Case Report: A 45-year-old woman with metastatic breast cancer was treated with bevacizumab and paclitaxel. The lung metastases were decreased but the meningitis carcinoma developed rapidly and she died. During the treatment with bevacizumab and paclitaxel, blood samples were taken serially and ctDNA was analyzed using a next-generation sequencer. TP53 frameshift mutation (TP53; p.Arg158fs with 7 nucleotides deletion) was identified in the tumor, and dynamic change in this mutation in ctDNA was observed in serially sampled plasma in this patient. We observed a rapid decrease of TP53 mutation at the beginning of treatment, then it increased as a sign of relapse. However, the high allelic fraction value of TP53 mutation was not consistent during the progression of cancer, suggesting that several factors affected the value of ctDNA. Conclusions: Although this is a single-case experience, it strongly suggests ctDNA could be a modality to determine the cancer status in real time. However, we found that several factors affected the value of ctDNA. Further investigations are needed to reveal the significance of these very high-sensitivity changes. PMID:28065930

  10. Investigation of the persistence of nerve agent degradation analytes on surfaces through wipe sampling and detection with ultrahigh performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Willison, Stuart A

    2015-01-20

    The persistence of chemical warfare nerve agent degradation analytes on surfaces is important, from indicating the presence of nerve agent on a surface to guiding environmental restoration of a site after a release. Persistence was investigated for several chemical warfare nerve agent degradation analytes on indoor surfaces and presents an approach for wipe sampling of surfaces, followed by wipe extraction and liquid chromatography-tandem mass spectrometry detection. Commercially available wipe materials were investigated to determine optimal wipe recoveries. Tested surfaces included porous/permeable (vinyl tile, painted drywall, and wood) and largely nonporous/impermeable (laminate, galvanized steel, and glass) surfaces. Wipe extracts were analyzed by ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). UPLC provides a separation of targeted degradation analytes in addition to being nearly four times faster than high-performance liquid chromatography, allowing for greater throughput after a large-scale contamination incident and subsequent remediation events. Percent recoveries from nonporous/impermeable surfaces were 60-103% for isopropyl methylphosphonate (IMPA), GB degradate; 61-91% for ethyl methylphosphonate (EMPA), VX degradate; and 60-98% for pinacolyl methylphosphonate (PMPA), GD degradate. Recovery efficiencies for methyl phosphonate (MPA), nerve agent degradate, and ethylhydrogen dimethylphosphonate (EHDMAP), GA degradate, were lower, perhaps due to matrix effects. Diisopropyl methylphosphonate, GB impurity, was not recovered from surfaces. The resulting detection limits for wipe extracts were 0.065 ng/cm(2) for IMPA, 0.079 ng/cm(2) for MPA, 0.040 ng/cm(2) for EMPA, 0.078 ng/cm(2) for EHDMAP, and 0.013 ng/cm(2) for PMPA. The data indicate that laboratories may hold wipe samples for up to 30 days prior to analysis. Target analytes were observed to persist on surfaces for at least 6 weeks.

  11. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    SciTech Connect

    Jackson, Christopher B.; Gallati, Sabina; Schaller, Andre

    2012-07-06

    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

  12. 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

    Holland, Mitchell M; Cave, Christopher A; Holland, Charity A; Bille, Todd W

    2003-06-01

    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

  13. Hematopoietic gene promoters subjected to a group-combinatorial study of DNA samples: identification of a megakaryocytic selective DNA signature

    PubMed Central

    Hazony, Yehonathan; Lu, Jun; St. Hilaire, Cynthia; Ravid, Katya

    2006-01-01

    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. PMID:16936310

  14. The Detection of Spotted Fever Group Rickettsia DNA in Tick Samples From Pastoral Communities in Kenya.

    PubMed

    Koka, Hellen; Sang, Rosemary; Kutima, Helen Lydia; Musila, Lillian

    2017-01-10

    In this study, ticks from pastoral communities in Kenya were tested for Rickettsia spp. infections in geographical regions where the presence of tick-borne arboviruses had previously been reported. Rickettsial and arbovirus infections have similar clinical features which makes differential diagnosis challenging when both diseases occur. The tick samples were tested for Rickettsia spp. by conventional PCR using three primer sets targeting the gltA, ompA, and ompB genes followed by amplicon sequencing. Of the tick pools screened, 25% (95/380) were positive for Rickettsia spp. DNA using the gltA primer set. Of the tick-positive pools, 60% were ticks collected from camels. Rickettsia aeschlimannii and R. africae were the main Rickettsia spp. detected in the tick pools sequenced. The findings of this study indicate that multiple Rickettsia species are circulating in ticks from pastoral communities in Kenya and could contribute to the etiology of febrile illness in these areas. Diagnosis and treatment of rickettsial infections should be a public health priority in these regions.

  15. An improved protocol for DNA extraction from alkaline soil and sediment samples for constructing metagenomic libraries.

    PubMed

    Verma, Digvijay; Satyanarayana, T

    2011-09-01

    An improved single-step protocol has been developed for extracting pure community humic substance-free DNA from alkaline soils and sediments. The method is based on direct cell lysis in the presence of powdered activated charcoal and polyvinylpolypyrrolidone followed by precipitation with polyethyleneglycol and isopropanol. The strategy allows simultaneous isolation and purification of DNA while minimizing the loss of DNA with respect to other available protocols for metagenomic DNA extraction. Moreover, the purity levels are significant, which are difficult to attain with any of the methods reported in the literature for DNA extraction from soils. The DNA thus extracted was free from humic substances and, therefore, could be processed for restriction digestion, PCR amplification as well as for the construction of metagenomic libraries.

  16. Genomic DNA isolation of Acrocomia aculeata (Arecaceae) from leaf and stipe tissue samples for PCR analysis.

    PubMed

    Lanes, E C M; Nick, C; Kuki, K N; Freitas, R D; Motoike, S Y

    2013-09-23

    Macaw palm, Acrocomia aculeata is an oleaginous species of the Arecaceae family; it has been identified as one of the most promising plants for sustainable production of renewable energy, especially biodiesel. We developed an efficient protocol of genomic DNA extraction for A. aculeata using leaf and stipe tissues, based on the cationic hexadecyltrimethylammonium bromide method, and we evaluated the quantity, purity, and integrity of the resultant DNA. We also determined whether these procedures interfere with PCR amplification using SSR molecular markers. The lowest concentration of DNA was obtained from stipe tissues (135 ng/μL), while fresh leaf tissues provided the highest concentration of DNA (650 ng/μL). Good quality DNA was obtained from fresh leaf, lyophilized leaf, and stipe tissues (relative purity, 1.79-1.89 nm). Differences in quantity and quality of DNA extracted from different tissues did not interfere with general patterns of PCR amplification based on SSR markers.

  17. Spectral, thermal, kinetic, molecular modeling and eukaryotic DNA degradation studies for a new series of albendazole (HABZ) complexes

    NASA Astrophysics Data System (ADS)

    El-Metwaly, Nashwa M.; Refat, Moamen S.

    2011-01-01

    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.

  18. Molding single DNA molecules in metals and sample preparation for electronic sequencing.

    PubMed

    Lund, John A; Parviz, Babak A

    2009-01-01

    We demonstrate the molding of single DNA molecules in 8 nm thin platinum molds. The molded structures have an apparent depth of 1 nm under STM imaging, and closely follow the contours of the DNA molecules. We have confirmed the presence of the embedded molecules and have verified the ability of this technique to scale down to single molecules. Additionally, we have utilized this method to perform electron tunneling analysis on embedded DNA molecules.

  19. Diagnostic accuracy of DNA methylation for head and neck cancer varies by sample type and number of markers tested

    PubMed Central

    Ji, Xu; Guan, Chao; Jiang, Xuejun; Li, Hong

    2016-01-01

    Abnormal methylation of certain cancer related genes strongly predicts a diagnosis of head and neck cancer (HNC), while the predictive power of methylation of other DNA markers for HNC remains unclear. To systemically assess the diagnostic value of DNA methylation patterns for HNC and the effect of methylation platform techniques and sample types, we performed a PubMed search for studies of the correlation between DNA methylation and HNC completed before July 2016, and extracted the sensitivity and specificity for methylated biomarkers. Across these studies, DNA methylation showed high sensitivity for diagnosing HNC in solid tissue (0.57), and high specificity in saliva (0.89). Area under the curve (AUC) from summary receiver operating characteristic (SROC) curves revealed that DNA methylation had more diagnostic power in solid tissue (AUC = 0.82) than saliva (AUC = 0.80) or blood (AUC = 0.77). Combinations of multiple methylated genes were more sensitive diagnostic markers than single methylated genes. Our results suggest that the diagnostic accuracy of methylated biomarkers for HNC varied by sample type and were most accurate when results from multiple sample types were considered. PMID:27683120

  20. Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq.

    PubMed

    Zhang, Zhian; Kermekchiev, Milko B; Barnes, Wayne M

    2010-03-01

    PCR-based clinical and forensic tests often have low sensitivity or even false-negative results caused by potent PCR inhibitors found in blood and soil. It is widely accepted that purification of target DNA before PCR is necessary for successful amplification. In an attempt to overcome PCR inhibition, enhance PCR amplification, and simplify the PCR protocol, we demonstrate improved PCR-enhancing cocktails containing nonionic detergent, l-carnitine, d-(+)-trehalose, and heparin. These cocktails, in combination with two inhibitor-resistant Taq mutants, OmniTaq and Omni Klentaq, enabled efficient amplification of exogenous, endogenous, and high-GC content DNA targets directly from crude samples containing human plasma, serum, and whole blood without DNA purification. In the presence of these enhancer cocktails, the mutant enzymes were able to tolerate at least 25% plasma, serum, or whole blood and as high as 80% GC content templates in PCR reactions. These enhancer cocktails also improved the performance of the novel Taq mutants in real-time PCR amplification using crude samples, both in SYBR Green fluorescence detection and TaqMan assays. The novel enhancer mixes also facilitated DNA amplification from crude samples with various commercial Taq DNA polymerases.

  1. Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding, genotyping, and disease diagnostics from fungal and oomycete samples.

    PubMed

    Osmundson, Todd W; Eyre, Catherine A; Hayden, Katherine M; Dhillon, Jaskirn; Garbelotto, Matteo M

    2013-01-01

    The ubiquity, high diversity and often-cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one-step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single-copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe-based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol-chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol-chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use.

  2. Simultaneous assessment of the macrobiome and microbiome in a bulk sample of tropical arthropods through DNA metasystematics

    PubMed Central

    Gibson, Joel; Shokralla, Shadi; Porter, Teresita M.; King, Ian; van Konynenburg, Steven; Janzen, Daniel H.; Hallwachs, Winnie; Hajibabaei, Mehrdad

    2014-01-01

    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

  3. Simultaneous assessment of the macrobiome and microbiome in a bulk sample of tropical arthropods through DNA metasystematics.

    PubMed

    Gibson, Joel; Shokralla, Shadi; Porter, Teresita M; King, Ian; van Konynenburg, Steven; Janzen, Daniel H; Hallwachs, Winnie; Hajibabaei, Mehrdad

    2014-06-03

    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.

  4. 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

    Brandfass, Christoph; Karlovsky, Petr

    2008-11-01

    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) an