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1

Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces  

PubMed Central

Background Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (?) can be estimated by determining the rate of decline. Results The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean ?predator = 0.0106 per nucleotide; mean ?prey = 0.0176 per nucleotide). This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples. Conclusion We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will allow researchers to measure template quality in order to evaluate alternate sources of DNA, different methods of sample preservation and different DNA extraction protocols. The technique could also be applied to study the process of DNA decay. PMID:16911807

Deagle, Bruce E; Eveson, J Paige; Jarman, Simon N

2006-01-01

2

A quantitative PCR assay for the assessment of DNA degradation in forensic samples  

Microsoft Academic Search

A multiplex quantitative PCR assay has been designed to amplify target sequences of different length, which allows for the assessment of DNA degradation in samples of forensic interest. The targets were chosen to provide quantification and fragment length information relevant to the STR amplification targets commonly used for forensic genotyping. The longer target (nuTH01, 170–190bp) spans the TH01 STR locus.

Katie L. Swango; Mark D. Timken; Mavis Date Chong; Martin R. Buoncristiani

2006-01-01

3

A quantitative PCR assay for the assessment of DNA degradation in forensic samples.  

PubMed

A multiplex quantitative PCR assay has been designed to amplify target sequences of different length, which allows for the assessment of DNA degradation in samples of forensic interest. The targets were chosen to provide quantification and fragment length information relevant to the STR amplification targets commonly used for forensic genotyping. The longer target (nuTH01, 170-190 bp) spans the TH01 STR locus. Although not one of the longest loci used for STR genotyping, it was chosen as a good compromise given the target length limitations on qPCR efficiency with TaqMan detection. The shorter target (nuCSF, 67 bp) was designed in the upstream flanking region of the CSF1PO STR locus. In addition to these human nuclear targets, the assay includes an internal PCR control target sequence to allow for an assessment of PCR inhibition. The assay was rigorously tested on samples with varying amounts of degradation, and the ratio of nuCSF:nuTH01 quantifications was shown to provide a good estimation of the degree of degradation present in a sample. This estimate, along with the internal control for PCR inhibition, provides a valuable tool for post-extraction sample assessment. PMID:15936161

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

2006-04-20

4

A duplex real-time qPCR assay for the quantification of human nuclear and mitochondrial DNA in forensic samples: implications for quantifying DNA in degraded samples.  

PubMed

A duplex real-time qPCR assay was developed for quantifying human nuclear and mitochondrial DNA in forensic samples. The nuclear portion of the assay utilized amplification of a approximately 170-190 bp target sequence that spans the repeat region of the TH01 STR locus, and the mitochondrial portion of the assay utilized amplification of a 69 bp target sequence in the ND1 region. Validation studies, performed on an ABI 7000 SDS instrument using TaqMan detection, demonstrated that both portions of the duplex assay provide suitable quantification sensitivity and precision down to 10-15 copies of each genome of interest and that neither portion shows cross-reactivity to commonly encountered non-human genomes. As part of the validation studies, a series of DNase-degraded samples were quantified using three different methods: the duplex nuclear-mitochondrial qPCR assay, the ABI Quantifiler Human DNA Quantification Kit qPCR assay, which amplifies and detects a 62 bp nuclear target sequence, and slot blot hybridization. For non-degraded and moderately degraded samples in the series, all three methods were suitably accurate for quantifying nuclear DNA to achieve successful STR amplifications to yield complete profiles using the ABI AmpFlSTR Identifiler kit. However, for highly degraded samples, the duplex qPCR assay provided better estimates of nuclear template for STR amplification than did either the commercial qPCR assay, which overestimated the quantity of STR-sized DNA fragments, leading to an increased proportion of undetected alleles at the larger STR loci, or slot blot hybridization, which underestimated the quantity of nuclear DNA, leading to an increased proportion of STR amplification artifacts due to amplification of excess template. PMID:16225209

Timken, Mark D; Swango, Katie L; Orrego, Cristián; Buoncristiani, Martin R

2005-09-01

5

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

PubMed Central

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. Multiple rounds of in-solution hybridisation-based DNA capture can retrieve whole mitochondrial genome sequences from even the most challenging samples. PMID:24289217

2013-01-01

6

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  

Microsoft Academic Search

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 ?170–190bp target sequence that spans the TH01 STR locus to quantify total human DNA (nuTH01), a 137bp target sequence directly adjacent to the SRY gene to quantify human male DNA (nuSRY),

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

2008-01-01

7

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

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) guidelines. PMID:19083806

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

2008-03-01

8

‘Mitominis’: multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples  

Microsoft Academic Search

The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the\\u000a two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300–400 bp\\u000a each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and\\u000a more stable approach using shortened

Cordula Eichmann; Walther Parson

2008-01-01

9

Shear degradation of DNA.  

PubMed Central

A concentric-cylinder flow-birefringence instrument is used to generate sufficient shear fields to break T2 DNA (M = 1.2 X 10(8)) and E. coli DNA (M = 2.5 X 10(9)) in dilute solution. Breakage is monitored in situ by measuring the change in birefringence relaxation after the flow has been stopped. The breakage of T2 DNA follows first-order kinetics. Rate constants are obtained as functions of shear rate and viscosity (varied by adding glycerol). The data are fitted by a modified Arrhenius equation, assuming that stess increases the rate by lowering the activation energy. The rate increases with temperature, pH, and water concentration, and appears to be a base-catalyzed hydrolysis of the phosphate-ester linkage. La3+ ions catalyze the reaction. E. coli DNA was reduced to half molecules at a shear stress of 0.4 dynes/cm2, which is about 2500 times less than that required for T2. The difference in rates is accounted for in part by the difference in size of the two, but may also reflect the presence of many single-strand nicks in the coli DNA. PMID:19729

Adam, R E; Zimm, B H

1977-01-01

10

Radiation-induced degradation of DNA bases  

Microsoft Academic Search

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

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

1999-01-01

11

STR analysis of degraded DNA using a miniplex  

Microsoft Academic Search

Analysis of short tandem repeat (STR) markers currently represents the most useful instrument in the field of forensic genetics. The problem with forensic material is the degradation of the sample material. In recent years, several papers have demonstrated that short amplicon STR (miniSTR) represents one of the most useful tools for analyzing degraded DNA samples.In the present study, we attempted

M. Nastainczyk; S. Schulz; M. Kleiber; U. D. Immel

2009-01-01

12

Persistent damage induces mitochondrial DNA degradation  

PubMed Central

Considerable progress has been made recently toward understanding the processes of mitochondrial DNA (mtDNA) damage and repair. However, a paucity of information still exists regarding the physiological effects of persistent mtDNA damage. This is due, in part, to experimental difficulties associated with targeting mtDNA for damage, while sparing nuclear DNA. Here, we characterize two systems designed for targeted mtDNA damage based on the inducible (Tet-ON) mitochondrial expression of the bacterial enzyme, exonuclease III, and the human enzyme, uracil-N-glyosylase containing the Y147A mutation. In both systems, damage was accompanied by degradation of mtDNA, which was detectable by six hours after induction of mutant uracil-N-glycosylase and by twelve hours after induction of exoIII. Unexpectedly, increases in the steady-state levels of single-strand lesions, which led to degradation, were small in absolute terms indicating that both abasic sites and single-strand gaps may be poorly tolerated in mtDNA. mtDNA degradation was accompanied by the loss of expression of mtDNA-encoded COX2. After withdrawal of the inducer, recovery from mtDNA depletion occurred faster in the system expressing exonuclease III, but in both systems reduced mtDNA levels persisted longer than 144h after doxycycline withdrawal. mtDNA degradation was followed by reduction and loss of respiration, decreased membrane potential, reduced cell viability, reduced intrinsic reactive oxygen species production, slowed proliferation, and changes in mitochondrial morphology (fragmentation of the mitochondrial network, rounding and “foaming” of the mitochondria). The mutagenic effects of abasic sites in mtDNA were low, which indicates that damaged mtDNA molecules may be degraded if not rapidly repaired. This study establishes, for the first time, that mtDNA degradation can be a direct and immediate consequence of persistent mtDNA damage and that increased ROS production is not an invariant consequence of mtDNA damage. PMID:23721969

Shokolenko, Inna N.; Wilson, Glenn L.; Alexeyev, Mikhail F.

2013-01-01

13

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

Microsoft Academic Search

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

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

14

Authentication of forensic DNA samples.  

PubMed

Over the past twenty years, DNA analysis has revolutionized forensic science, and has become a dominant tool in law enforcement. Today, DNA evidence is key to the conviction or exoneration of suspects of various types of crime, from theft to rape and murder. However, the disturbing possibility that DNA evidence can be faked has been overlooked. It turns out that standard molecular biology techniques such as PCR, molecular cloning, and recently developed whole genome amplification (WGA), enable anyone with basic equipment and know-how to produce practically unlimited amounts of in vitro synthesized (artificial) DNA with any desired genetic profile. This artificial DNA can then be applied to surfaces of objects or incorporated into genuine human tissues and planted in crime scenes. Here we show that the current forensic procedure fails to distinguish between such samples of blood, saliva, and touched surfaces with artificial DNA, and corresponding samples with in vivo generated (natural) DNA. Furthermore, genotyping of both artificial and natural samples with Profiler Plus((R)) yielded full profiles with no anomalies. In order to effectively deal with this problem, we developed an authentication assay, which distinguishes between natural and artificial DNA based on methylation analysis of a set of genomic loci: in natural DNA, some loci are methylated and others are unmethylated, while in artificial DNA all loci are unmethylated. The assay was tested on natural and artificial samples of blood, saliva, and touched surfaces, with complete success. Adopting an authentication assay for casework samples as part of the forensic procedure is necessary for maintaining the high credibility of DNA evidence in the judiciary system. PMID:20129467

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

2010-02-01

15

Survival and DNA degradation in anhydrobiotic tardigrades.  

PubMed

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

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

2009-12-01

16

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

PubMed

Analysis of short tandem repeat makers has become the most powerful tool for DNA typing in forensic casework analysis. Unfortunately, typing of DNA extracted from telogen shed hairs, bones buried in the soil or from paraffin-embedded, formalin-fixed tissue often reveals no results due to the degradation of DNA. The reduction in size of the target fragments by development of new primers and their combination in multiplex approaches open a new field of DNA analysis. Here we present a new sensitive short pentaplex PCR including the loci amelogenin, TH01, VWA, D3S1358 and D8S1179. Validation tests of our new method included sensitivity, mixtures, human specificity, artificial degradation of DNA by DNase I and case work analysis on a panel of different forensic samples. The detection limit was 12.5 pg of human DNA, and mixtures of 50 pg in a total of 1000 pg were clearly detectable and revealed complete profiles. Only DNA extracts of human primates displayed a few signals, whereas other animal, fungal or bacterial DNA showed no signals. Our method proved extremely valuable in the analysis of artificially degraded DNA and in forensic cases, where only poorly preserved DNA was available. This approach and other similar methods can aid in the analysis of samples where allelic drop out of larger fragments is observed. It is highly recommended to develop more of these multiplexes to improve poor quality DNA typing. PMID:16814503

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

2007-03-01

17

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...

18

Degradation of DNA by nucleases in intestinal tract of rats.  

PubMed

Strains of Escherichia coli K12 have been constructed as safer hosts for use in recombinant DNA research, These strains are unable to survive passage through the intestinal tracts of rats because of a constellation of mutations conferring bile sensitivity and requirements for diaminopimelic acid and thymine. Since death caused by diaminopimelic acid deprivation could release recombinant DNA before DNA is degraded because of thymine starvation, it is important to determine the "survival potential" of the released DNA's. Bacterial and plasmid DNA's extracted from bacterial cells are rapidly degraded when added to low dilutions of rat intestinal contents. This observation, coupled with the stringent requirements necessary for in vitro transformation or transfection, make in vivo transmission of naked recombinant DNA in the rat intestinal tract highly improbable. PMID:322286

Maturin, L; Curtiss, R

1977-04-01

19

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

NASA Astrophysics Data System (ADS)

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

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

2005-05-01

20

Magnetic Field Exposure Induces DNA Degradation  

Microsoft Academic Search

In our earlier experiments, we discovered that magnetic field exposure could bring both stabilizing and destabilizing effects to the DNA of Escherichia coli, depending on our parameters of assessment, and both of these effects were associated with the induced synthesis of the heat shock proteins Hsp70\\/Hsp40 (DnaK\\/DnaJ). These contradicting results prompted us to explore in this study the effect of

Shun Hau Li; King-Chuen Chow

2001-01-01

21

Evaluating DNA degradation rates in faecal pellets of the endangered pygmy rabbit.  

PubMed

Noninvasive genetic sampling of faecal pellets can be a valuable method for monitoring rare and cryptic wildlife populations, like the pygmy rabbit (Brachylagus idahoensis). To investigate this method's efficiency for pygmy rabbit monitoring, we evaluated the effect of sample age on DNA degradation in faecal pellets under summer field conditions. We placed 275 samples from known individuals in natural field conditions for 1-60 days and assessed DNA quality by amplifying a 294-base-pair (bp) mitochondrial DNA (mtDNA) locus and five nuclear DNA (nDNA) microsatellite loci (111-221 bp). DNA degradation was influenced by sample age, DNA type, locus length and rabbit sex. Both mtDNA and nDNA exhibited high PCR success rates (94.4%) in samples <1 day old. Success rates for microsatellite loci declined rapidly from 80.0% to 42.7% between days 5 and 7, likely due to increased environmental temperature. Success rates for mtDNA amplification remained higher than nDNA over time, with moderate success (66.7%) at 21 days. Allelic dropout rates were relatively high (17.6% at <1 day) and increased to 100% at 60 days. False allele rates ranged from 0 to 30.0% and increased gradually over time. We recommend collecting samples as fresh as possible for individual identification during summer field conditions. Our study suggests that this method can be useful for future monitoring efforts, including occupancy surveys, individual identification, population estimation, parentage analysis and monitoring of genetic diversity both of a re-introduced population in central Washington and across their range. PMID:23590236

DeMay, Stephanie M; Becker, Penny A; Eidson, Chad A; Rachlow, Janet L; Johnson, Timothy R; Waits, Lisette P

2013-07-01

22

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

PubMed Central

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

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

2015-01-01

23

KLF6 degradation after apoptotic DNA damage.  

PubMed

Krüppel-like factor 6 (KLF6) is a cancer gene (). Here, we demonstrate that KLF6 protein is rapidly degraded when apoptosis is induced via the intrinsic pathway by cisplatin, adriamycin, or UVB irradiation in multiple cell lines (HCT116, SW40, HepG2, PC3-M, Skov3, NIH-3T3, 293T, GM09706, and MEF, IMR-90). KLF6 degradation occurred in the presence or absence of p53, was associated with ubiquitination, mediated by the proteasome (half-life 16min, unstimulated), and independent of caspases and calpain. KLF6 was unchanged by apoptosis via the extrinsic/death-receptor pathway. Deregulation of KLF6 stability may alter its tumor suppressor function and/or the response of tumors to chemotherapeutics. PMID:17113081

Banck, Michaela S; Beaven, Simon W; Narla, Goutham; Walsh, Martin J; Friedman, Scott L; Beutler, Andreas S

2006-12-22

24

APPLICATION OF DNA-DNA COLONY HYBRIDIZATION TO THE DETECTION OF CATABOLIC GENOTYPES IN ENVIRONMENTAL SAMPLES  

EPA Science Inventory

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, pLAFRI and RSF101...

25

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

PubMed Central

Anaerobic methyl tert-butyl ether (MTBE) degradation potential was investigated in samples from a range of sources. From these 22 experimental variations, only one source (from wastewater treatment plant samples) exhibited MTBE degradation. These microcosms were methanogenic and were subjected to DNA-based stable isotope probing (SIP) targeted to both bacteria and archaea to identify the putative MTBE degraders. For this purpose, DNA was extracted at two time points, subjected to ultracentrifugation, fractioning, and terminal restriction fragment length polymorphism (TRFLP). In addition, bacterial and archaeal 16S rRNA gene clone libraries were constructed. The SIP experiments indicated bacteria in the phyla Firmicutes (family Ruminococcaceae) and Alphaproteobacteria (genus Sphingopyxis) were the dominant MTBE degraders. Previous studies have suggested a role for Firmicutes in anaerobic MTBE degradation; however, the putative MTBE-degrading microorganism in the current study is a novel MTBE-degrading phylotype within this phylum. Two archaeal phylotypes (genera Methanosarcina and Methanocorpusculum) were also enriched in the heavy fractions, and these organisms may be responsible for minor amounts of MTBE degradation or for the uptake of metabolites released from the primary MTBE degraders. Currently, limited information exists on the microorganisms able to degrade MTBE under anaerobic conditions. This work represents the first application of DNA-based SIP to identify anaerobic MTBE-degrading microorganisms in laboratory microcosms and therefore provides a valuable set of data to definitively link identity with anaerobic MTBE degradation. PMID:22327600

Sun, Weimin; Sun, Xiaoxu

2012-01-01

26

Differential effect of sample preservation methods on plant and arbuscular mycorrhizal fungal DNA.  

PubMed

A wide range of methods are commonly used for preserving environmental samples prior to molecular analyses. However, the effect of these preservation methods on fungal DNA is not understood. The objective of this study was to test the effect of eight different preservation methods on the quality and yield of DNA extracted from Bromus inermis and Daucus carota roots colonized by the arbuscular mycorrhizal (AM) fungus, Glomus intraradices. The total DNA concentration in sample extracts was quantified using spectrophotometry. Samples that were frozen (-80 masculineC and -20 masculineC), stored in 95% ethanol, or silica gel dried yielded total (plant and fungal) DNA concentrations that were not significantly different from fresh samples. In contrast, samples stored in CTAB solution or freeze-dried resulted in significantly reduced DNA concentrations compared with fresh samples. The preservation methods had no effect on the purity of the sample extracts for both plant species. However, the DNA of the dried samples (silica gel dried, freeze-dried, heat dried) appeared to be slightly more degraded compared with samples that remained hydrated (frozen, stored in ethanol or CTAB solutions) during storage when visualized on a gel. The concentration of AM fungal DNA in sample extracts was quantified using TaqMan real time PCR. Methods that preserved samples in hydrated form had similar AM fungal DNA concentrations as fresh samples, except D. carota samples stored in ethanol. In contrast, preservation methods that involved drying the samples had very low concentrations of AM fungal DNA for B. inermis, and nearly undetectable for D. carota samples. The drying process appears to be a major factor in the degradation of AM fungal DNA while having less of an impact on plant DNA. Based on these results, samples that need to be preserved prior to molecular analysis of AM fungi should be kept frozen to minimize the degradation of plant and AM fungal DNA. PMID:20470836

Bainard, L D; Klironomos, J N; Hart, M M

2010-08-01

27

DNA Compaction Induced by a Cationic Polymer or Surfactant Impact Gene Expression and DNA Degradation  

PubMed Central

There is an increasing interest in achieving gene regulation in biotechnological and biomedical applications by using synthetic DNA-binding agents. Most studies have so far focused on synthetic sequence-specific DNA-binding agents. Such approaches are relatively complicated and cost intensive and their level of sophistication is not always required, in particular for biotechnological application. Our study is inspired by in vivo data that suggest that DNA compaction might contribute to gene regulation. This study exploits the potential of using synthetic DNA compacting agents that are not sequence-specific to achieve gene regulation for in vitro systems. The semi-synthetic in vitro system we use include common cationic DNA-compacting agents, poly(amido amine) (PAMAM) dendrimers and the surfactant hexadecyltrimethylammonium bromide (CTAB), which we apply to linearized plasmid DNA encoding for the luciferase reporter gene. We show that complexing the DNA with either of the cationic agents leads to gene expression inhibition in a manner that depends on the extent of compaction. This is demonstrated by using a coupled in vitro transcription-translation system. We show that compaction can also protect DNA against degradation in a dose-dependent manner. Furthermore, our study shows that these effects are reversible and DNA can be released from the complexes. Release of DNA leads to restoration of gene expression and makes the DNA susceptible to degradation by Dnase. A highly charged polyelectrolyte, heparin, is needed to release DNA from dendrimers, while DNA complexed with CTAB dissociates with the non-ionic surfactant C12E5. Our results demonstrate the relation between DNA compaction by non-specific DNA-binding agents and gene expression and gene regulation can be achieved in vitro systems in a reliable dose-dependent and reversible manner. PMID:24671109

Ainalem, Marie-Louise; Bartles, Andrew; Muck, Joscha; Dias, Rita S.; Carnerup, Anna M.; Zink, Daniele; Nylander, Tommy

2014-01-01

28

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

NASA Astrophysics Data System (ADS)

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 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. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr12005d

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

2012-03-01

29

Characterization of New MiniSTR Loci to Aid Analysis of Degraded DNA  

Microsoft Academic Search

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

Michael D. Coble; John M. Butler

2005-01-01

30

Automatable full demineralization DNA extraction procedure from degraded skeletal remains.  

PubMed

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

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

2012-05-01

31

DNA Fingerprinting by Sampled Sequencing  

Microsoft Academic Search

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

Sydney Brenner; Kenneth J. Livak

1989-01-01

32

Microfluidic DNA sample preparation method and device  

DOEpatents

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

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

2002-01-01

33

Post Mortem DNA Degradation of Human Tissue Experimentally Mummified in Salt  

PubMed Central

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

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

2014-01-01

34

Post mortem DNA degradation of human tissue experimentally mummified in salt.  

PubMed

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

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

2014-01-01

35

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

PubMed

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. PMID:23870938

Ballari, Rajashekhar V; Martin, Asha

2013-12-01

36

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

PubMed

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

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

2014-01-01

37

Flow cytometric detection method for DNA samples  

DOEpatents

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

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

2006-08-01

38

Flow cytometric detection method for DNA samples  

DOEpatents

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

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

2011-07-05

39

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2013 CFR

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

2013-07-01

40

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2010 CFR

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

2010-07-01

41

28 CFR 28.12 - Collection of DNA samples.  

...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 §...

2014-07-01

42

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2012 CFR

...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 §...

2012-07-01

43

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2011 CFR

...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 §...

2011-07-01

44

Effects of hypoxanthine substitution on bleomycin-mediated DNA strand degradation in DNA-RNA hybrids.  

PubMed Central

We have reported on the differences in site-specific cleavage between DNA and DNA-RNA hybrids by various prototypic DNA cleavers (accompanying paper). In the case of bleomycin (BLM), degradation at 5'-GC-3'sites was suppressed relative to the same sequence in double-stranded DNA, while 5'-GT-3' damage remained constant. We now present results of our further investigation on the chemical and conformational factors that contribute to BLM-mediated DNA strand cleavage of DNA-RNA hybrids. Substitution of guanine by hypoxanthine on the RNA strand of hybrids resulted in a significant enhancement of 5'-GC-3' site damage on the DNA strand relative to double-stranded DNA, thus reversing the suppression noted at these sites. Additionally, 5'-AT-3' sites, which are damaged significantly more in the hybrid than in DNA, exhibit decreased product formation when hypoxanthine is present on the RNA strand of hybrids. However, when hypoxanthine is substituted for guanine on the DNA strand (a GC cleavage site becomes IC), 5'-IT-3' and 5'-IC-3' site cleavage is almost completely suppressed, whereas AT site cleavage is dramatically enhanced. The priority in metallobleomycin site-specific cleavage of hybrids changes with hypoxanthine substitution: the cleavage priority is AT > GT > GC in native hybrid; GC > GT > AT in hybrids substituted with hypoxanthine in the RNA strand; AT >> GT approximately GC in hybrids substituted with hypoxanthine in the DNA strand. The results of kinetic isotope effect studies on BLM cleavage are presented and, in most cases, the values are larger for the hypoxanthine-substituted hybrid. The results suggest that the 2-amino groups of guanine residues on both strands of the nucleic acid play an important role in modulation of the binding and cleavage specificity of BLM. PMID:9108170

Bansal, M; Stubbe, J; Kozarich, J W

1997-01-01

45

Degradation of methylmercury by bacteria isolated from environmental samples.  

PubMed

A total of 207 bacterial cultures, isolated from environmental samples, was screened for ability to degrade methylmercury. Of these, 30 were found positive for aerobic demethylation. Twenty-two of these were shown to be facultative anaerobes and 21 of these degraded methylmercury anaerobically. All positive species volatilized methylmercury aerobically, and methane was produced as a degradation product. Although methylmercury degradation was complete in most cases, material balances indicated some of the inorganic mercury formed was not volatilized and is presumed bound to the cells. All positive isolates were tolerant to at least 0.5 mug of methylmercury per ml, and the extent of volatilization of mercury increased with concentration to the threshold value. The results indicate that demethylating species are prevalent in the environment and may be important in suppressing the methylmercury content of sediments. PMID:4572979

Spangler, W J; Spigarelli, J L; Rose, J M; Flippin, R S; Miller, H H

1973-04-01

46

FBXL5-mediated degradation of single-stranded DNA-binding protein hSSB1 controls DNA damage response.  

PubMed

Human single-strand (ss) DNA binding proteins 1 (hSSB1) has been shown to participate in DNA damage response and maintenance of genome stability by regulating the initiation of ATM-dependent signaling. ATM phosphorylates hSSB1 and prevents hSSB1 from ubiquitin-proteasome-mediated degradation. However, the E3 ligase that targets hSSB1 for destruction is still unknown. Here, we report that hSSB1 is the bona fide substrate for an Fbxl5-containing SCF (Skp1-Cul1-F box) E3 ligase. Fbxl5 interacts with and targets hSSB1 for ubiquitination and degradation, which could be prevented by ATM-mediated hSSB1 T117 phosphorylation. Furthermore, cells overexpression of Fbxl5 abrogated the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets and exhibited increased radiosensitivity, chemosensitivity and defective checkpoint activation after genotoxic stress stimuli. Moreover, the protein levels of hSSB1 and Fbxl5 showed an inverse correlation in lung cancer cells lines and clinical lung cancer samples. Therefore, Fbxl5 may negatively modulate hSSB1 to regulate DNA damage response, implicating Fbxl5 as a novel, promising therapeutic target for lung cancers. PMID:25249620

Chen, Zhi-Wei; Liu, Bin; Tang, Nai-Wang; Xu, Yun-Hua; Ye, Xiang-Yun; Li, Zi-Ming; Niu, Xiao-Min; Shen, Sheng-Ping; Lu, Shun; Xu, Ling

2015-01-01

47

Determination of the early time of death by computerized image analysis of DNA degradation: Which is the best quantitative indicator of DNA degradation?  

Microsoft Academic Search

Summary  This study evaluated the correlation between DNA degradation of the splenic lymphocytes and the early time of death, examined\\u000a the early time of death by computerized image analysis technique (CIAT) and identified the best parameter that quantitatively\\u000a reflects the DNA degradation. The spleen tissues from 34 SD rats were collected, subjected to cell smearing every 2 h within\\u000a the first

Lijiang Liu; Xiji Shu; Liang Ren; Hongyan Zhou; Yan Li; Wei Liu; Cheng Zhu; Liang Liu

2007-01-01

48

Metagenomics: DNA sequencing of environmental samples  

SciTech Connect

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

Tringe, Susannah Green; Rubin, Edward M.

2005-09-01

49

Mitochondrial DNA analysis of Swedish population samples.  

PubMed

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

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

2013-11-01

50

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

PubMed Central

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

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

2012-01-01

51

Collecting and preserving biological samples from challenging environments for DNA analysis.  

PubMed

Biological materials collected in harsh environments such as archaeological excavations, at crime scenes, after mass disasters, in museums, or non-invasively in the field constitute a highly valuable source of genetic information. However, poor quality and limited quantity of the DNA extracted from these samples can be extremely challenging during further analyses. Here we have reviewed how degradation, decomposition, and contamination can affect DNA analysis, and how correct sample collection and storage methods will ensure the best possible conditions for further genetic analysis. Furthermore, highly efficient protocols for collection, decontamination, and extraction of DNA from minute amounts of biological material are presented. PMID:24620766

Bu?, Magdalena M; Allen, Marie

2014-02-01

52

Chromosomal lesion suppression and removal in Escherichia coli via linear DNA degradation.  

PubMed Central

RecBCD is a DNA helicase/exonuclease implicated in degradation of foreign linear DNA and in RecA-dependent recombinational repair of chromosomal lesions in E. coli. The low viability of recA recBC mutants vs. recA mutants indicates the existence of RecA-independent roles for RecBCD. To distinguish among possible RecA-independent roles of the RecBCD enzyme in replication, repair, and DNA degradation, we introduced wild-type and mutant combinations of the recBCD chromosomal region on a low-copy-number plasmid into a DeltarecA DeltarecBCD mutant and determined the viability of resulting strains. Our results argue against ideas that RecBCD is a structural element in the replication factory or is involved in RecA-independent repair of chromosomal lesions. We found that RecBCD-catalyzed DNA degradation is the only activity important for the recA-independent viability, suggesting that degradation of linear tails of sigma-replicating chromosomes could be one of the RecBCD's roles. However, since the weaker DNA degradation capacity due a combination of the RecBC helicase and ssDNA-specific exonucleases restores viability of the DeltarecA DeltarecBCD mutant to a significant extent, we favor suppression of chromosomal lesions via linear DNA degradation at reversed replication forks as the major RecA-independent role of the RecBCD enzyme. PMID:12702673

Miranda, Anabel; Kuzminov, Andrei

2003-01-01

53

XPB mediated retroviral cDNA degradation coincides with entry to the nucleus  

SciTech Connect

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

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

2011-02-20

54

Waikato DNA Sequencing Facility Please send samples to  

E-print Network

Waikato DNA Sequencing Facility Please send samples to: Attention: Waikato DNA Sequencing Facility: ............................................... Dilution Factor: ................................ Template Name Template Type (ss/dsDNA or PCR product pairs) Primer Name Primer Conc. Please supply at 5pmol/uL DNA Purification Method

Waikato, University of

55

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

PubMed

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 be useful for restoring STR profiles from damaged DNA, but further work is required to develop a generalized approach. PMID:24997322

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

56

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

PubMed

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. PMID:24243191

Mutou, Chiaki; Tanaka, Katsunori; Ishikawa, Ryuji

2014-01-01

57

Sliding Window Analyses for Optimal Selection of Mini-Barcodes, and Application to 454-Pyrosequencing for Specimen Identification from Degraded DNA  

PubMed Central

DNA barcoding remains a challenge when applied to diet analyses, ancient DNA studies, environmental DNA samples and, more generally, in any cases where DNA samples have not been adequately preserved. Because the size of the commonly used barcoding marker (COI) is over 600 base pairs (bp), amplification fails when the DNA molecule is degraded into smaller fragments. However, relevant information for specimen identification may not be evenly distributed along the barcoding region, and a shorter target can be sufficient for identification purposes. This study proposes a new, widely applicable, method to compare the performance of all potential ‘mini-barcodes’ for a given molecular marker and to objectively select the shortest and most informative one. Our method is based on a sliding window analysis implemented in the new R package SPIDER (Species IDentity and Evolution in R). This method is applicable to any taxon and any molecular marker. Here, it was tested on earthworm DNA that had been degraded through digestion by carnivorous landsnails. A 100 bp region of 16 S rDNA was selected as the shortest informative fragment (mini-barcode) required for accurate specimen identification. Corresponding primers were designed and used to amplify degraded earthworm (prey) DNA from 46 landsnail (predator) faeces using 454-pyrosequencing. This led to the detection of 18 earthworm species in the diet of the snail. We encourage molecular ecologists to use this method to objectively select the most informative region of the gene they aim to amplify from degraded DNA. The method and tools provided here, can be particularly useful (1) when dealing with degraded DNA for which only small fragments can be amplified, (2) for cases where no consensus has yet been reached on the appropriate barcode gene, or (3) to allow direct analysis of short reads derived from massively parallel sequencing without the need for bioinformatic consolidation. PMID:22666489

Boyer, Stephane; Brown, Samuel D. J.; Collins, Rupert A.; Cruickshank, Robert H.; Lefort, Marie-Caroline; Malumbres-Olarte, Jagoba; Wratten, Stephen D.

2012-01-01

58

Automatable full demineralization DNA extraction procedure from degraded skeletal remains  

Microsoft Academic Search

During the 7 year period from 2002 to 2009 a high volume, silica-binding DNA extraction protocol for bone, based on modified QIAGEN's Blood Maxi Kit protocol was highly successful permitting the DNA matching of >14,500 missing persons from former Yugoslavia. This method, however, requires large amount of bone material and large volumes of reagents. The logical evolution was to develop

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

59

Interpretation of the Apollo 14 Thermal Degradation Sample experiment  

NASA Astrophysics Data System (ADS)

The Thermal Degradation Sample (TDS) experiment was one of the many investigations performed on the lunar surface during Apollo 14. Remarkably, the results of this 40 year old experiment were never fully interpreted, perhaps in part because the hardware vanished after its return. Mission records, high resolution photographs returned from the mission, and recent laboratory investigations have been used to glean important results from this experiment. It is most likely that the dust adhesion to the TDS was less than anticipated because of atomic-level contamination of its surfaces. These contaminants were probably removed from most equipment surfaces on the Moon by sputter cleaning by the solar wind, but the TDS experiments were not exposed to the solar wind long enough to affect the cleaning.

Gaier, James R.

2012-09-01

60

Bacterial and fungal DNA extraction from blood samples: automated protocols.  

PubMed

Automation in DNA isolation is a necessity for routine practice employing molecular diagnosis of infectious agents. To this end, the development of automated systems for the molecular diagnosis of microorganisms directly in blood samples is at its beginning. Important characteristics of systems demanded for routine use include high recovery of microbial DNA, DNA-free containment for the reduction of DNA contamination from exogenous sources, DNA-free reagents and consumables, ideally a walkaway system, and economical pricing of the equipment and consumables. Such full automation of DNA extraction evaluated and in use for sepsis diagnostics is yet not available. Here, we present protocols for the semiautomated isolation of microbial DNA from blood culture and low- and high-volume blood samples. The protocols include a manual pretreatment step followed by automated extraction and purification of microbial DNA. PMID:25319785

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

2015-01-01

61

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)

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

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

2002-01-01

62

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

PubMed Central

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. PMID:23144869

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

2012-01-01

63

Wet Lab: DNA Barcoding: From Samples to Sequences  

NSDL National Science Digital Library

This is a lab activity outlined by a PDF and accompanied by a PowerPoint presentation to be shown to students. Students perform the wet lab experiments necessary for DNA barcoding. Students will purify DNA, perform polymerase chain reaction (PCR), and analyze the PCR products by agarose gel electrophoresis. This resource is listed at the bottom of the lessons on the URL referenced, and titled "Wet Lab: DNA Barcoding: From Samples to Sequences."

Jodie Spitze (Kent Meridian High School)

2011-04-01

64

Determination of the early time of death by computerized image analysis of DNA degradation: which is the best quantitative indicator of DNA degradation?  

PubMed

This study evaluated the correlation between DNA degradation of the splenic lymphocytes and the early time of death, examined the early time of death by computerized image analysis technique (CIAT) and identified the best parameter that quantitatively reflects the DNA degradation. The spleen tissues from 34 SD rats were collected, subjected to cell smearing every 2 h within the first 36 h after death, stained by Feulgen-Van's staining, three indices reflecting DNA content in splenic lymphocytes, including integral optical density (IOD), average optical density (AOD), average gray scale (AG) were measured by the image analysis. Our results showed that IOD and AOD decreased and AG increased over time within the first 36 h. A stepwise linear regression analysis showed that only AG was fitted. A correlation between the postmortem interval (PMI) and AG was identified and the corresponding regression equation was obtained. Our study suggests that CIAT is a useful and promising tool for the estimation of early PMI with good objectivity and reproducibility, and AG is a more effective and better quantitative indicator for the estimation of PMI within the first 36 h after death in rats. PMID:17828487

Liu, Lijiang; Shu, Xiji; Ren, Liang; Zhou, Hongyan; Li, Yan; Liu, Wei; Zhu, Cheng; Liu, Liang

2007-08-01

65

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

PubMed Central

A number of recent improvements have provided contemporary forensic investigations with a variety of tools to improve the analysis of mixed DNA samples in criminal investigations, producing notable improvements in the analysis of complex trace samples in cases of sexual assult and homicide. Mixed DNA contains DNA from two or more contributors, compounding DNA analysis by combining DNA from one or more major contributors with small amounts of DNA from potentially numerous minor contributors. These samples are characterized by a high probability of drop-out or drop-in combined with elevated stutter, significantly increasing analysis complexity. At some loci, minor contributor alleles may be completely obscured due to amplification bias or over-amplification, creating the illusion of additional contributors. Thus, estimating the number of contributors and separating contributor genotypes at a given locus is significantly more difficult in mixed DNA samples, requiring the application of specialized protocols that have only recently been widely commercialized and standardized. Over the last decade, the accuracy and repeatability of mixed DNA analyses available to conventional forensic laboratories has greatly advanced in terms of laboratory technology, mathematical models and biostatistical software, generating more accurate, rapid and readily available data for legal proceedings and criminal cases. PMID:24748965

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

2014-01-01

66

Investigating bacterial populations in styrene-degrading biofilters by 16S rDNA tag pyrosequencing.  

PubMed

Microbial biofilms are essential components in the elimination of pollutants within biofilters, yet still little is known regarding the complex relationships between microbial community structure and biodegradation function within these engineered ecosystems. To further explore this relationship, 16S rDNA tag pyrosequencing was applied to samples taken at four time points from a styrene-degrading biofilter undergoing variable operating conditions. Changes in microbial structure were observed between different stages of biofilter operation, and the level of styrene concentration was revealed to be a critical factor affecting these changes. Bacterial genera Azoarcus and Pseudomonas were among the dominant classified genera in the biofilter. Canonical correspondence analysis (CCA) and correlation analysis revealed that the genera Brevundimonas, Hydrogenophaga, and Achromobacter may play important roles in styrene degradation under increasing styrene concentrations. No significant correlations (P?>?0.05) could be detected between biofilter operational/functional parameters and biodiversity measurements, although biological heterogeneity within biofilms and/or technical variability within pyrosequencing may have considerably affected these results. Percentages of selected bacterial taxonomic groups detected by fluorescence in situ hybridization (FISH) were compared to results from pyrosequencing in order to assess the effectiveness and limitations of each method for identifying each microbial taxon. Comparison of results revealed discrepancies between the two methods in the detected percentages of numerous taxonomic groups. Biases and technical limitations of both FISH and pyrosequencing, such as the binding of FISH probes to non-target microbial groups and lack of classification of sequences for defined taxonomic groups from pyrosequencing, may partially explain some differences between the two methods. PMID:24950754

Portune, Kevin J; Pérez, M Carmen; Álvarez-Hornos, F Javier; Gabaldón, Carmen

2015-01-01

67

Sequencing the hypervariable regions of human mitochondrial DNA using massively parallel sequencing: Enhanced data acquisition for DNA samples encountered in forensic testing.  

PubMed

Mitochondrial DNA testing is a useful tool in the analysis of forensic biological evidence. In cases where nuclear DNA is damaged or limited in quantity, the higher copy number of mitochondrial genomes available in a sample can provide information about the source of a sample. Currently, Sanger-type sequencing (STS) is the primary method to develop mitochondrial DNA profiles. This method is laborious and time consuming. Massively parallel sequencing (MPS) can increase the amount of information obtained from mitochondrial DNA samples while improving turnaround time by decreasing the numbers of manipulations and more so by exploiting high throughput analyses to obtain interpretable results. In this study 18 buccal swabs, three different tissue samples from five individuals, and four bones samples from casework were sequenced at hypervariable regions I and II using STS and MPS. Sample enrichment for STS and MPS was PCR-based. Library preparation for MPS was performed using Nextera® XT DNA Sample Preparation Kit and sequencing was performed on the MiSeq™ (Illumina, Inc.). MPS yielded full concordance of base calls with STS results, and the newer methodology was able to resolve length heteroplasmy in homopolymeric regions. This study demonstrates short amplicon MPS of mitochondrial DNA is feasible, can provide information not possible with STS, and lays the groundwork for development of a whole genome sequencing strategy for degraded samples. PMID:25459369

Davis, Carey; Peters, Dixie; Warshauer, David; King, Jonathan; Budowle, Bruce

2014-10-25

68

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

PubMed Central

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

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

2010-01-01

69

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

PubMed

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

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

2010-07-15

70

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

PubMed

A magnetoresistive biosensing platform based on a single magnetic tunnel junction (MTJ) scanning probe and DNA microarrays labeled with magnetic particles has been developed to provide an inexpensive, sensitive and reliable detection of DNA. The biosensing platform was demonstrated on a DNA microarray assay for quantifying bacteria capable of degrading methyl tertiary butyl ether (MTBE), where concentrations as low as 10 pM were detectable. Synthetic probe bacterial DNA was immobilized on a microarray glass slide surface, hybridized with the 48 base pair long biotinylated target DNA and subsequently incubated with streptavidin-coated 2.8 ?m diameter magnetic particles. The biosensing platform then makes use of a micron-sized MTJ sensor that was raster scanned across a 3 mm by 5 mm glass slide area to capture the stray magnetic field from the tagged DNA and extract two dimensional magnetic field images of the microarray. The magnetic field output is then averaged over each 100 ?m diameter DNA array spot to extract the magnetic spot intensity, analogous to the fluorescence spot intensity used in conventional optical scanners. The magnetic scanning result is compared with results from a commercial laser scanner and particle coverage optical counting to demonstrate the dynamic range and linear sensitivity of the biosensing platform as a potentially inexpensive, sensitive and portable alternative for DNA microarray detection for field applications. PMID:20889328

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

2011-01-15

71

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

PubMed Central

A magnetoresistive biosensing platform based on a single magnetic tunnel junction (MTJ) scanning probe and DNA microarrays labeled with magnetic particles has been developed to provide an inexpensive, sensitive and reliable detection of DNA. The biosensing platform was demonstrated on a DNA microarray assay for quantifying bacteria capable of degrading methyl tertiary-butyl ether (MTBE), where concentrations as low as 10 pM were detectable. Synthetic probe bacterial DNA was immobilized on a microarray glass slide surface, hybridized with the 48 base pair long biotinylated target DNA and subsequently incubated with streptavidin-coated 2.8 ?m diameter magnetic particles. The biosensing platform then makes use of a micron-sized MTJ sensor that was raster scanned across a 3 mm by 5 mm glass slide area to capture the stray magnetic field from the tagged DNA and extract two dimensional magnetic field images of the microarray. The magnetic field output is then averaged over each 100 ?m diameter DNA array spot to extract the magnetic spot intensity, analogous to the fluorescence spot intensity used in conventional optical scanners. The magnetic scanning result is compared with results from a commercial laser scanner and particle coverage optical counting to demonstrate the dynamic range and linear sensitivity of the biosensing platform as a potentially inexpensive, sensitive and portable alternative for DNA microarray detection for field applications. PMID:20889328

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

2010-01-01

72

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

PubMed

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

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

2013-11-01

73

A DNA methylation fingerprint of 1628 human samples  

PubMed Central

Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases. PMID:21613409

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

2012-01-01

74

Improved reproducibility in genome-wide DNA methylation analysis for PAXgene-fixed samples compared with restored formalin fixation and paraffin-embedding DNA.  

PubMed

Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin fixation and paraffin-embedding (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap-freezing is the PAXgene Tissue System, developed for simultaneous preservation of morphology, proteins, and nucleic acids. In the current study, we compared the performance of DNA from either PAXgene or formalin-fixed tissues to snap-frozen material for genome-wide DNA methylation analysis using the Illumina 450K BeadChip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better compared with restored FFPE DNA in genome-wide DNA methylation analysis. In addition, DNA from PAXgene tissue can be directly used on the array without prior restoration, rendering the analytical process significantly more time- and cost-effective. PMID:25277813

Andersen, Gitte Brinch; Hager, Henrik; Hansen, Lise Lotte; Tost, Jörg

2014-09-30

75

Valosin-containing protein regulates the proteasome-mediated degradation of DNA-PKcs in glioma cells.  

PubMed

DNA-dependent protein kinase (DNA-PK) has an important role in the repair of DNA damage and regulates the radiation sensitivity of glioblastoma cells. The VCP (valosine-containing protein), a chaperone protein that regulates ubiquitin-dependent protein degradation, is phosphorylated by DNA-PK and recruited to DNA double-strand break sites to regulate DNA damage repair. However, it is not clear whether VCP is involved in DNA-PKcs (DNA-PK catalytic subunit) degradation or whether it regulates the radiosensitivity of glioblastoma. Our data demonstrated that DNA-PKcs was ubiquitinated and bound to VCP. VCP knockdown resulted in the accumulation of the DNA-PKcs protein in glioblastoma cells, and the proteasome inhibitor MG132 synergised this increase. As expected, this increase promoted the efficiency of DNA repair in several glioblastoma cell lines; in turn, this enhanced activity decreased the radiation sensitivity and prolonged the survival fraction of glioblastoma cells in vitro. Moreover, the VCP knockdown in glioblastoma cells reduced the survival time of the xenografted mice with radiation treatment relative to the control xenografted glioblastoma mice. In addition, the VCP protein was also downregulated in ~25% of GBM tissues from patients (WHO, grade IV astrocytoma), and the VCP protein level was correlated with patient survival (R(2)=0.5222, P<0.05). These findings demonstrated that VCP regulates DNA-PKcs degradation and increases the sensitivity of GBM cells to radiation. PMID:23722536

Jiang, N; Shen, Y; Fei, X; Sheng, K; Sun, P; Qiu, Y; Larner, J; Cao, L; Kong, X; Mi, J

2013-01-01

76

28 CFR 28.13 - Analysis and indexing of DNA samples.  

Code of Federal Regulations, 2011 CFR

... 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 §...

2011-07-01

77

28 CFR 28.13 - Analysis and indexing of DNA samples.  

... 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 §...

2014-07-01

78

28 CFR 28.13 - Analysis and indexing of DNA samples.  

Code of Federal Regulations, 2012 CFR

... 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 §...

2012-07-01

79

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

Federal Register 2010, 2011, 2012, 2013

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

2010-06-07

80

28 CFR 28.13 - Analysis and indexing of DNA samples.  

Code of Federal Regulations, 2013 CFR

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

2013-07-01

81

28 CFR 28.13 - Analysis and indexing of DNA samples.  

Code of Federal Regulations, 2010 CFR

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

2010-07-01

82

A DNA microarray system for forensic SNP analysis  

Microsoft Academic Search

Forensic DNA analysis is routinely performed using polymorphic short tandem repeat (STR) markers. However, for degraded or minute DNA samples, analysis of autosomal single nucleotide polymorphisms (SNPs) in short fragments might be more successful. Furthermore, sequencing of mitochondrial DNA (mtDNA) is often performed on highly degraded or scarce samples due to the high copy number of mtDNA in each cell.

Anna-Maria Divne; Marie Allen

2005-01-01

83

Degradation of DNA damage-independently stalled RNA polymerase II is independent of the E3 ligase Elc1  

PubMed Central

Transcription elongation is a highly dynamic and discontinuous process, which includes frequent pausing of RNA polymerase II (RNAPII). RNAPII complexes that stall persistently on a gene during transcription elongation block transcription and thus have to be removed. It has been proposed that the cellular pathway for removal of these DNA damage-independently stalled RNAPII complexes is similar or identical to the removal of RNAPII complexes stalled due to DNA damage. Here, we show that—consistent with previous data—DNA damage-independent stalling causes polyubiquitylation and proteasome-mediated degradation of Rpb1, the largest subunit of RNAPII, using Saccharomyces cerevisiae as model system. Moreover, recruitment of the proteasome to RNAPII and transcribed genes is increased when transcription elongation is impaired indicating that Rpb1 degradation takes place at the gene. Importantly, in contrast to the DNA damage-dependent pathway Rpb1 degradation of DNA damage-independently stalled RNAPII is independent of the E3 ligase Elc1. In addition, deubiquitylation of RNAPII is also independent of the Elc1-antagonizing deubiquitylase Ubp3. Thus, the pathway for degradation of DNA damage-independently stalled RNAPII is overlapping yet distinct from the previously described pathway for degradation of RNAPII stalled due to DNA damage. Taken together, we provide the first evidence that the cell discriminates between DNA damage-dependently and -independently stalled RNAPII. PMID:25120264

Karakasili, Eleni; Burkert-Kautzsch, Cornelia; Kieser, Anja; Sträßer, Katja

2014-01-01

84

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

E-print Network

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 ...

Adiconis, Xian

85

Validation of DNA methylation profiling in formalin-fixed paraffin-embedded samples using the Infinium HumanMethylation450 Microarray.  

PubMed

A formalin-fixed paraffin-embedded (FFPE) sample usually yields highly degraded DNA, which limits the use of techniques requiring high-quality DNA, such as Infinium Methylation microarrays. To overcome this restriction, we have applied an FFPE restoration procedure consisting of DNA repair and ligation processes in a set of paired fresh-frozen (FF) and FFPE samples. We validated the FFPE results in comparison with matched FF samples, enabling us to use FFPE samples on the Infinium HumanMethylation450 Methylation array. PMID:24732293

Moran, Sebastián; Vizoso, Miguel; Martinez-Cardús, Anna; Gomez, Antonio; Matías-Guiu, Xavier; Chiavenna, Sebastián M; Fernandez, Andrés G; Esteller, Manel

2014-06-01

86

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

PubMed Central

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

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

2014-01-01

87

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

PubMed

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

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

2014-01-01

88

Ancient DNA studies: new perspectives on old samples.  

PubMed

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

Rizzi, Ermanno; Lari, Martina; Gigli, Elena; De Bellis, Gianluca; Caramelli, David

2012-01-01

89

Preamplification Procedure for the Analysis of Ancient DNA Samples  

PubMed Central

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

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

2013-01-01

90

Preamplification procedure for the analysis of ancient DNA samples.  

PubMed

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

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

2013-01-01

91

A comparison of five methods for extracting DNA from paucicellular clinical samples  

Microsoft Academic Search

Translational protocols in cancer and carcinogenesis often require isolation of genomic DNA from paucicellular clinical samples. DNA extraction methods for PCR-based applications should optimize the recovery of amplifiable DNA. We compared five methods for DNA extraction in paucicellular epithelial and lymphocyte samples using proportion of extractions producing amplifiable DNA and mean real-time PCR Ct values for GAPDH as the endpoint

Leslie Cler; Dawei Bu; Cheryl Lewis; David Euhus

2006-01-01

92

Crystal Structure of CRN-4: Implications for Domain Function in Apoptotic DNA Degradation?  

PubMed Central

Cell death related nuclease 4 (CRN-4) is one of the apoptotic nucleases involved in DNA degradation in Caenorhabditis elegans. To understand how CRN-4 is involved in apoptotic DNA fragmentation, we analyzed CRN-4's biochemical properties, in vivo cell functions, and the crystal structures of CRN-4 in apo-form, Mn2+-bound active form, and Er3+-bound inactive form. CRN-4 is a dimeric nuclease with the optimal enzyme activity in cleaving double-stranded DNA in apoptotic salt conditions. Both mutational studies and the structures of the Mn2+-bound CRN-4 revealed the geometry of the functional nuclease active site in the N-terminal DEDDh domain. The C-terminal domain, termed the Zn-domain, contains basic surface residues ideal for nucleic acid recognition and is involved in DNA binding, as confirmed by deletion assays. Cell death analysis in C. elegans further demonstrated that both the nuclease active site and the Zn-domain are required for crn-4's function in apoptosis. Combining all of the data, we suggest a structural model where chromosomal DNA is bound at the Zn-domain and cleaved at the DEDDh nuclease domain in CRN-4 when the cell is undergoing apoptosis. PMID:18981218

Hsiao, Yu-Yuan; Nakagawa, Akihisa; Shi, Zhonghao; Mitani, Shohei; Xue, Ding; Yuan, Hanna S.

2009-01-01

93

Leishmania DNA is rapidly degraded following parasite death: An analysis by microscopy and real-time PCR.  

E-print Network

the 120 h time period studied correlated with Leishmania DNA as quantified by real-time PCR. Our results1 Leishmania DNA is rapidly degraded following parasite death: An analysis by microscopy and real-time in experimental models involves PCR-based assays. Compared to time-consuming conventional methods, this type

Paris-Sud XI, Université de

94

Evaluation of methods that subdue the effects of polymerase chain reaction inhibitors in the study of ancient and degraded DNA  

E-print Network

Evaluation of methods that subdue the effects of polymerase chain reaction inhibitors in the study Keywords: Ancient DNA Degraded DNA Polymerase chain reaction inhibitors Polymerases Species identification the potential for inhibiting the polymerase chain reaction (PCR), the means by which minute amounts of genetic

Kemp, Brian M.

95

Extra-bodily DNA sampling by the police.  

PubMed

Forensic investigators have statutory powers to take DNA samples directly from suspects' bodies in certain circumstances but sometimes the powers fall short, legally or practically Police may then look for samples that have become separated from their suspects for one reason or another. No jurisdiction currently bars or even regulates this practice, which is instead loosely governed by laws on property, consent and evidence. This article argues that this lack of regulation undermines the entire system of forensic procedure laws. PMID:24597386

Gans, Jeremy

2013-12-01

96

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

PubMed

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

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

2013-07-01

97

A REVIEW OF CURRENT KNOWLEDGE OF TECHNIQUES TO EXTRACT AND AMPLIFY DNA FROM 'DIFFICULT' WHALE SAMPLES  

Microsoft Academic Search

DNA analysis methods currently used for whale product identification are dependent on extraction and PCR amplification of cetacean nucleic acids, but certain product types and intensive processing may restrict the amount of DNA recovered or degrade the DNA and inhibit amplification. Newly developed methods developed for \\

Frank Cipriano; Luis Pastene

2009-01-01

98

Degradation of mutant initiator protein DnaA204 by proteases ClpP, ClpQ and Lon is prevented when DNA is SeqA-free.  

PubMed Central

A mutant form of the Escherichia coli replication initiator protein, DnaA204, is unstable. At low growth rates, the dnaA204 mutant cells experience a limitation of initiator protein and grow with reduced initiation frequency and DNA concentration. The mutant DnaA protein is stabilized by the lack of SeqA protein. This stabilization was also observed in a dam mutant where the chromosome remains unmethylated. Since unmethylated DNA is not bound by SeqA, this indicates that DnaA204 is not stabilized by the lack of SeqA protein by itself, but rather by lack of SeqA complexed with DNA. Thus the destabilization of DnaA204 may be due either to interaction with SeqA-DNA complexes or changes in nucleoid organization and superhelicity caused by SeqA. The DnaA204 protein was processed through several chaperone/protease pathways. The protein was stabilized by the presence of the chaperones ClpA and ClpX and degraded by their cognate protease ClpP. The dnaA204 mutant was not viable in the absence of ClpY, indicating that this chaperone is essential for DnaA204 stability or function. Its cognate protease ClpQ, as well as Lon protease, degraded DnaA204 to the same degree as ClpP. The chaperones GroES, GroEL and DnaK contributed to stabilization of DnaA204 protein. PMID:12479794

Slominska, Monika; Wahl, Anne; Wegrzyn, Grzegorz; Skarstad, Kirsten

2003-01-01

99

Optimization of kinetic parameters for the degradation of plasmid DNA in rat plasma  

NASA Astrophysics Data System (ADS)

Biotechnology is a rapidly growing area of research work in the field of pharmaceutical sciences. The study of pharmacokinetics of plasmid DNA (pDNA) is an important area of research work. It has been observed that the process of gene delivery faces many troubles on the transport of pDNA towards their target sites. The topoforms of pDNA has been termed as super coiled (S-C), open circular (O-C) and linear (L), the kinetic model of which will be presented in this paper. The kinetic model gives rise to system of ordinary differential equations (ODEs), the exact solution of which has been found. The kinetic parameters, which are responsible for the degradation of super coiled, and the formation of open circular and linear topoforms have a great significance not only in vitro but for modeling of further processes as well, therefore need to be addressed in great detail. For this purpose, global optimization techniques have been adopted, thus finding the optimal results for the said model. The results of the model, while using the optimal parameters, were compared against the measured data, which gives a nice agreement.

Chaudhry, Q. A.

2014-12-01

100

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

Federal Register 2010, 2011, 2012, 2013

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

2011-11-23

101

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

Federal Register 2010, 2011, 2012, 2013

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

2012-06-11

102

Improved Methods of Carnivore Faecal Sample Preservation, DNA Extraction and Quantification for Accurate Genotyping of Wild Tigers  

PubMed Central

Background Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase. Methods We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval. Results Average DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (p<0.001). Maximum DNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p<0.1 and p<0.001). This difference was not significant when samples were preserved by two-step method (p>0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively. Conclusions Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols. PMID:23071624

Harika, Katakam; Mahla, Ranjeet Singh; Shivaji, Sisinthy

2012-01-01

103

DNA Barcoding: Error Rates Based on Comprehensive Sampling  

PubMed Central

DNA barcoding has attracted attention with promises to aid in species identification and discovery; however, few well-sampled datasets are available to test its performance. We provide the first examination of barcoding performance in a comprehensively sampled, diverse group (cypraeid marine gastropods, or cowries). We utilize previous methods for testing performance and employ a novel phylogenetic approach to calculate intraspecific variation and interspecific divergence. Error rates are estimated for (1) identifying samples against a well-characterized phylogeny, and (2) assisting in species discovery for partially known groups. We find that the lowest overall error for species identification is 4%. In contrast, barcoding performs poorly in incompletely sampled groups. Here, species delineation relies on the use of thresholds, set to differentiate between intraspecific variation and interspecific divergence. Whereas proponents envision a “barcoding gap” between the two, we find substantial overlap, leading to minimal error rates of ~17% in cowries. Moreover, error rates double if only traditionally recognized species are analyzed. Thus, DNA barcoding holds promise for identification in taxonomically well-understood and thoroughly sampled clades. However, the use of thresholds does not bode well for delineating closely related species in taxonomically understudied groups. The promise of barcoding will be realized only if based on solid taxonomic foundations. PMID:16336051

2005-01-01

104

DNA degradation by aqueous extract of Aloe vera in the presence of copper ions.  

PubMed

The plant Aloe vera has long been used in medicine, as dietary supplements and for cosmetic purposes. Aloe vera extracts are a rich source of polyphenols, such as aloin and aloe emodin and have shown a wide range of pharmacological properties, including anti-inflammatory and anti-cancer properties. The bioactive component aloe emodin has been reported to induce apoptosis in various cancer cell lines. Many of the biological activities of Aloe vera have been attributed to its antioxidant properties. However, most plant-derived polyphenols that are also present in Aloe vera may exhibit pro-oxidant properties either alone or in the presence of transition metals, such as copper. Previous reports from this laboratory have implicated the pro-oxidant action as one of the mechanisms for their anti-cancer properties. In the present paper, we show that aqueous extract of Aloe vera is also able to cause DNA degradation in the presence of copper ions. Further, the extract is also able to reduce Cu(II) to Cu(I) and generate reactive oxygen species, such as superoxide anion and hydroxyl radicals in a dose-dependent manner, which correlates with ability of the extract to cause DNA breakage. Thus, the study shows that in addition to antioxidant activity, Aloe vera extract also possess pro-oxidant properties, leading to oxidative DNA breakage. PMID:20653287

Naqvi, Shoa; Ullah, M F; Hadi, S M

2010-06-01

105

Bacterial Argonaute samples the transcriptome to identify foreign DNA  

PubMed Central

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

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

2013-01-01

106

Flow cytofluorometric assay of human whole blood leukocyte DNA degradation in response to Yersinia pestis and Staphylococcus aureus  

NASA Astrophysics Data System (ADS)

Human leukocytes containing less than 2C DNA per cell (damaged or dead cells) were detected and quantified by flow cytometry and DNA-specific staining with ethidium bromide and mithramycin in whole blood infected with Staphylococcus aureus or Yersinia pestis. Addition of live S. aureus to the blood (100 microbe cells per one leukocyte) resulted in rapid degradation of leukocyte DNA within 3 to 6 hours of incubation at 37 degree(s)C. However, only about 50 percent cells were damaged and the leukocytes with the intact genetic apparatus could be found in the blood for a period up to 24 hours. The leukocyte injury was preceded by an increase of DNA per cell content (as compared to the normal one) that was likely to be connected with the active phagocytosis of S. aureus by granulocytes (2C DNA of diploid phagocytes plus the all bacterial DNA absorbed). In response to the same dose of actively growing (at 37 degree(s)C) virulent Y. pestis cells, no increase in DNA content per cell could be observed in the human blood leukocytes. The process of the leukocyte DNA degradation started after a 6-hour incubation, and between 18 to 24 hours of incubation about 90 percent leukocytes (phagocytes and lymphocytes) lost their specific DNA fluorescence. These results demonstrated a high potential of flow cytometry in comparative analysis in vitro of the leukocyte DNA degradation process in human blood in response to bacteria with various pathogenic properties. They agree with the modern idea of an apoptotic mechanism of immunosuppression in plague.

Kravtsov, Alexander L.; Grebenyukova, Tatyana P.; Bobyleva, Elena V.; Golovko, Elena M.; Malyukova, Tatyana A.; Lyapin, Mikhail N.; Kostyukova, Tatyana A.; Yezhov, Igor N.; Kuznetsov, Oleg S.

2001-05-01

107

Thermal degradation of weldable poly(vinyl chloride) samples at low temperatures.  

PubMed

A study was performed to determine possible emission products during plastic welding that may be responsible for health problems. Thermal degradation of poly(vinyl chloride) samples was carried out at 170 degrees C in a thermal desorption gas chromatography (GC) injector and in a modified impinger in a GC oven, in combination with various analytical techniques, thermogravimetric analysis, capillary GC, GC-mass spectrometry, high-performance liquid chromatography and isotachophoresis. Some of the degradation products found are known to be eye irritants. PMID:3215984

Andersson, B

1988-07-22

108

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

PubMed

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. PMID:20457058

Bowden, Anna; Fleming, Rachel; Harbison, SallyAnn

2011-01-01

109

Determination of DNase activity by degradation of ethidium bromide-DNA complexes using a fluorescence plate reader.  

PubMed

The long known toxicity of free chromatin mediated by histones regained attention after discovery of neutrophil extracellular traps (NETs). Free histones from necrotic cells or NETs can damage prokaryotic and eukaryotic cells and are responsible for the aggravation of a growing list of diseases. DNases degrade the toxic chromatin polymer to nucleosomes and efficiently reduce local high histone concentrations. Therefore, DNase activity as a biomarker is of growing interest in basic and clinical research. Here a detailed one-step protocol is presented that allows rapid and sensitive detection of DNases down to 400fg/?l per reaction based on the detection of fluorescent ethidium bromide/DNA complexes in a 96-well plate reader. The flexible protocol uses an internal standard for background correction and allows convenient and reliable data analysis using common laboratory equipment and chemicals without elaborate preparations. The DNase activity of a sample is clearly defined by substrate amount, incubation time, and (if appropriate) a DNase standard for absolute quantification in Kunitz units per milligram sample protein. Quantitative kinetic determination is possible within less than 1h down to 5pg DNases/?l per reaction. PMID:25433147

Vogel, Benjamin; Frantz, Stefan

2015-02-15

110

The Effect of Geographical Scale of Sampling on DNA Barcoding  

PubMed Central

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

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

2012-01-01

111

The effect of geographical scale of sampling on DNA barcoding.  

PubMed

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

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

2012-10-01

112

Comparison of DNA preservation methods for environmental bacterial community samples.  

PubMed

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. PMID:22974342

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

2013-02-01

113

Comparison of DNA preservation methods for environmental bacterial community samples  

USGS Publications Warehouse

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

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

2013-01-01

114

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

Microsoft Academic Search

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

Anna Bowden; Rachel Fleming; SallyAnn Harbison

2011-01-01

115

A Simple, Efficient Method for the Separation of Humic Substances and DNA from Environmental Samples  

PubMed Central

Three different gels (Sepharose 4B, Sephadex G-200, and Sephadex G-50) were evaluated as a means of removing humic contaminants from DNA extracts of environmental samples. Sepharose 4B gave superior separation of DNA from humics, and DNA purified in this way showed consistently greater amplification than DNA purified by the other materials. PMID:16535760

Jackson, C. R.; Harper, J. P.; Willoughby, D.; Roden, E. E.; Churchill, P. F.

1997-01-01

116

Nondestructive Sampling of Human Skeletal Remains Yields Ancient Nuclear and Mitochondrial DNA  

E-print Network

Nondestructive Sampling of Human Skeletal Remains Yields Ancient Nuclear and Mitochondrial DNA of Archaeology and Ethnology, Harvard University, Cambridge, MA 02138 KEY WORDS ancient DNA; nondestructive DNA to valuable specimens, we describe a nondestructive method for extracting DNA from ancient human remains

Kemp, Brian M.

117

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

PubMed

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

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

118

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

PubMed Central

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

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

119

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

PubMed Central

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

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

1986-01-01

120

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

Microsoft Academic Search

The selective degradation of mutated mitochondrial DNA (mtDNA) molecules is a potential strategy to re-populate cells with wild-type (wt) mtDNA mole- cules and thereby alleviate the defective mitochon- drial function that underlies mtDNA diseases. Zinc finger nucleases (ZFNs), which are nucleases con- jugated to a zinc-finger peptide (ZFP) engineered to bind a specific DNA sequence, could be useful for the

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

2008-01-01

121

ORIGINAL PAPER Evaluating the impact of non-lethal DNA sampling on two  

E-print Network

from the US Fish and Wildlife Service to sample DNA from N. m. mitchellii populations. We suggestORIGINAL PAPER Evaluating the impact of non-lethal DNA sampling on two butterflies, Vanessa cardui not be desirable or permitted. We set out to develop a demonstrably non-lethal method of obtaining DNA from

Landis, Doug

122

Structural Insights into Apoptotic DNA Degradation by CED-3 Protease Suppressor-6 (CPS-6) from Caenorhabditis elegans*  

PubMed Central

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 Mg2+-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 ? resolution, revealing a mixed ?? topology with the two ???-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 Mg2+-bound active site. Mutations of four aromatic and basic residues: Phe122, Arg146, Arg156, and Phe166, 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. PMID:22223640

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.

2012-01-01

123

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

USGS Publications Warehouse

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

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

2002-01-01

124

Comparison of DNA extraction methods for PCR amplification of mitochondrial cytochrome c oxidase subunit II (COII) DNA from primate fecal samples  

Microsoft Academic Search

Mitochondrial COII DNA was amplified by PCR from total DNA extracted from field collected primate fecal samples (n=24) which had been stored without refrigeration for over 30 days. High molecular weight DNA total DNA was obtained from samples stored in 70% (v\\/v) ethanol, SDS lysis buffer (LB) and guanidine isothiocyanate buffer (GTB) than from samples stored in 10% formalin. Fecal

Christopher A. Whittier; Arun K. Dhar; Chip Stem; Jane Goodall; Acacia Alcivar-Warren

1999-01-01

125

Applications of pooled DNA samples to the assessment of population affinities: Short Tandem Repeats (STR)  

E-print Network

Pooled DNA samples have been used in association studies of Mendelian disease genes. This method involves combining equal quantities of DNA from patients and control subjects into separate pools and comparing the pools for distributions of genetic...

Crawford, Michael H.; Banerjee, P.; Demarchi, D. A.; Zlojutro, Mark; McComb, J.; Livshits, Gregory; Henneberg, M.; Mosher, M. J.; Schanfield, M. S.; Knowles, J. A.

2005-01-01

126

The successful recovery of low copy number and degraded DNA from bones exposed to seawater suitable for generating a DNA STR profile.  

PubMed

A universal method allowing for DNA profiling from bones exposed to seawater has not been reported yet. This study refers on the identification of a body immersed in seawater for 8 months. The biological material for identification was the mandibular body, usually characterized by low success rates of DNA analysis. Initially, two extraction protocols were performed with negative results: one used for bones immersed in fresh water and a silica-column procedure. A third protocol was performed, which combined the extraction of a higher amount of bone powder, the use of multi-silica-based extraction columns followed by a concentration step. This protocol allowed to obtain low copy number DNA and to generate a 12-loci STR profile by combining conventional STR typing and mini-STR technologies. This protocol could be suitable when human bones have been exposed to severe environmental conditions, and the available nuclear DNA is highly degraded and in low copy number. PMID:24313323

Mameli, Alessandro; Piras, Gavino; Delogu, Giovanni

2014-03-01

127

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

PubMed Central

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

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

2015-01-01

128

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

PubMed

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

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

2015-01-01

129

Protocol for Optimal Quality and Quantity Pollen DNA Isolation from Honey Samples  

PubMed Central

The present study illustrates an optimized sample preparation method for an efficient DNA isolation from low quantities of honey samples. A conventional PCR-based method was validated, which potentially enables characterization of plant species from as low as 3 ml bee-honey samples. In the present study, an anionic detergent was used to lyse the hard outer pollen shell, and DTT was used for isolation of thiolated DNA, as it might facilitate protein digestion and assists in releasing the DNA into solution, as well as reduce cross-links between DNA and other biomolecules. Optimization of both the quantity of honey sample and time duration for DNA isolation was done during development of this method. With the use of this method, chloroplast DNA was successfully PCR amplified and sequenced from honey DNA samples. PMID:25365793

Lalhmangaihi, Ralte; Ghatak, Souvik; Laha, Ramachandra; Gurusubramanian, Guruswami; Kumar, Nachimuthu Senthil

2014-01-01

130

CRN-1, a Caenorhabditis elegans FEN-1 homologue, cooperates with CPS-6/EndoG to promote apoptotic DNA degradation  

PubMed Central

Oligonucleosomal fragmentation of chromosomes in dying cells is a hallmark of apoptosis. Little is known about how it is executed or what cellular components are involved. We show that crn-1, a Caenorhabditis elegans homologue of human flap endonuclease-1 (FEN-1) that is normally involved in DNA replication and repair, is also important for apoptosis. Reduction of crn-1 activity by RNA interference resulted in cell death phenotypes similar to those displayed by a mutant lacking the mitochondrial endonuclease CPS-6/endonuclease G. CRN-1 localizes to nuclei and can associate and cooperate with CPS-6 to promote stepwise DNA fragmentation, utilizing the endonuclease activity of CPS-6 and both the 5?–3? exonuclease activity and a previously uncharacterized gap-dependent endonuclease activity of CRN-1. Our results suggest that CRN-1/FEN-1 may play a critical role in switching the state of cells from DNA replication/repair to DNA degradation during apoptosis. PMID:12840007

Parrish, Jay Z.; Yang, Chonglin; Shen, Binghui; Xue, Ding

2003-01-01

131

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

PubMed Central

Ciliates are unicellular eukaryotic organisms containing two types of nuclei: macronuclei and micronuclei. After the sexual pathway takes place, a new macronucleus is formed from a zygote nucleus, whereas the old macronucleus is degraded and resorbed. In the course of macronuclear differentiation, polytene chromosomes are synthesized that become degraded again after some hours. Most of the DNA is eliminated, and the remaining DNA is fragmented into small DNA molecules that are amplified to a high copy number in the new macronucleus. The protein Pdd1p (programmed DNA degradation protein 1) from Tetrahymena has been shown to be present in macronuclear anlagen in the DNA degradation stage and also in the old macronuclei, which are resorbed during the formation of the new macronucleus. In this study the identification and localization of a Pdd1p homologous protein in Stylonychia (Spdd1p) is described. Spdd1p is localized in the precursor nuclei in the DNA elimination stage and in the old macronuclei during their degradation, but also in macronuclei and micronuclei of starved cells. In all of these nuclei, apoptotic-like DNA breakdown was detected. These data suggest that Spdd1p is a general factor involved in programmed DNA degradation in Stylonychia. PMID:10473642

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

1999-01-01

132

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

PubMed Central

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

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

133

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

Microsoft Academic Search

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

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

2006-01-01

134

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

PubMed Central

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

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

2008-01-01

135

EFFECTIVE METHOD TO EXTRACT DNA FROM ENVIRONMENTAL SAMPLES FOR POLYMERASE CHAIN REACTION AMPLIFICATION AND DNA FINGERPRINT ANALYSIS  

EPA Science Inventory

A rapid direct-extraction method was used to obtain DNA from environmental soil samples. eat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. he DNA was purified by agarose gel electrophoresis, amplified with 16S based primers by use of the polymerase chain rea...

136

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

PubMed

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

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

2014-07-01

137

A model for sample stacking in microcapillary DNA electrophoresis  

E-print Network

Sanger's method of chain termination is the method of choice in DNA sequencing, where electrophoresis is used to separate the different sized DNA. In the past decade, microfabricated capillary devices have been developed ...

Srivastava, Alok Kumar, 1967-

2002-01-01

138

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

PubMed

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. PMID:17673223

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

2007-09-14

139

Solid supported in situ derivatization extraction of acidic degradation products of nerve agents from aqueous samples.  

PubMed

This study deals with the solid supported in situ derivatization extraction of acidic degradation products of nerve agents present in aqueous samples. Target analytes were alkyl alkylphosphonic acids and alkylphosphonic acids, which are important environmental signatures of nerve agents. The method involved tert-butyldimethylchlorosilane mediated in situ silylation of analytes on commercially available diatomaceous solid phase extraction cartridges. Various parameters such as derivatizing reagent, its concentration, reaction time, temperature and eluting solvent were optimized. Recoveries of the analytes were determined by GC-MS which ranged from 60% to 86%. The limits of detection (LOD) and limit of quantification (LOQ) with selected analytes were achieved down to 78 and 213ngmL(-1) respectively, in selected ion monitoring mode. The successful applicability of method was also demonstrated on samples of biological origin such as plasma and to the samples received in 34th official proficiency test conducted by the Organization for Prohibition the of Chemical Weapons. PMID:25103280

Chinthakindi, Sridhar; Purohit, Ajay; Singh, Varoon; Tak, Vijay; Dubey, D K; Pardasani, Deepak

2014-09-12

140

The Pinpoint DNA Isolation System as a novel DNA sampling method in forensic biology  

Microsoft Academic Search

The Pinpoint DNA Isolation System (Zymo Research) uses a dissolved polymer compound to remove and extract DNA from slide mounted pathology specimens. This polymer is applied to a non-porous substrate on which biological material is deposited, allowed to dry, and the polymer, containing cells and DNA, is peeled off. The polymer dissolves into solution during extraction, theoretically releasing more DNA

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

141

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

PubMed

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

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

2013-12-01

142

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

USGS Publications Warehouse

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

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

2013-01-01

143

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

PubMed

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

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

2012-02-13

144

IDENTIFICATION OF IN SITU 2,4-DICHLOROPHENOXYACETIC ACID-DEGRADING SOIL MICROORGANISMS USING DNA-STABLE ISOTOPE PROBING  

Technology Transfer Automated Retrieval System (TEKTRAN)

Stable isotope probing (SIP) was used to investigate the microorganisms responsible for degradation of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) in soil samples. Soils were unamended or amended with either unlabelled 2,4-D or UL(ring) 13C-2,4-D. Removal of 2,4-D was complete after 17 day...

145

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

PubMed

Exposure of bacterial cells to honey inhibits their growth and may cause cell death. Our previous studies showed a cause-effect relationship between hydroxyl radical generated from honey hydrogen peroxide and growth arrest. Here we explored the role of hydroxyl radicals as inducers of bacterial cells death. The bactericidal effect of ·OH on antibiotic-resistant clinical isolates of MRSA and VRE and standard bacterial strains of E. coli and B. subtiles was examined using a broth microdilution assay supplemented with 3'-(p-aminophenyl) fluorescein (APF) as the ·OH trap, followed by colony enumeration. Bactericidal activities of eight honeys (six varieties of buckwheat, blueberry and manuka honeys) were analyzed. The MBC/MIC ratio ?4 and the killing curves indicated that honeys exhibited powerful, concentration-dependent bactericidal effect. The extent of killing depended on the ratio of honey concentration to bacterial load, indicating that honey dose was critical for its bactericidal efficacy. The killing rate and potency varied between honeys and ranged from over a 6-log(10) to 4-log(10) CFU/ml reduction of viable cells, equivalent to complete bacterial eradication. The maximal killing was associated with the extensive degradation of bacterial DNA. Honey concentration at which DNA degradation occurred correlated with cell death observed in the concentration-dependent cell-kill on agar plates. There was no quantitative relationship between the ·OH generation by honey and bactericidal effect. At the MBC, where there was no surviving cells and no DNA was visible on agarose gels, the ·OH levels were on average 2-3x lower than at Minimum Inhibitory Concentration (MICs) (p < 0.0001). Pre-treatment of honey with catalase, abolished the bactericidal effect. This raised possibilities that either the abrupt killing prevented accumulation of ·OH (dead cells did not generate ·OH) or that DNA degradation and killing is the actual footprint of ·OH action. In conclusion, honeys of buckwheat origin exhibited powerful, concentration-dependent bactericidal effect. The killing and DNA degradation showed a cause-effect relationship. Hydrogen peroxide was an active part of honey killing mechanism. PMID:22783246

Brudzynski, Katrina; Abubaker, Kamal; Wang, Tony

2012-01-01

146

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

PubMed Central

Exposure of bacterial cells to honey inhibits their growth and may cause cell death. Our previous studies showed a cause-effect relationship between hydroxyl radical generated from honey hydrogen peroxide and growth arrest. Here we explored the role of hydroxyl radicals as inducers of bacterial cells death. The bactericidal effect of ·OH on antibiotic-resistant clinical isolates of MRSA and VRE and standard bacterial strains of E. coli and B. subtiles was examined using a broth microdilution assay supplemented with 3?-(p-aminophenyl) fluorescein (APF) as the ·OH trap, followed by colony enumeration. Bactericidal activities of eight honeys (six varieties of buckwheat, blueberry and manuka honeys) were analyzed. The MBC/MIC ratio ?4 and the killing curves indicated that honeys exhibited powerful, concentration-dependent bactericidal effect. The extent of killing depended on the ratio of honey concentration to bacterial load, indicating that honey dose was critical for its bactericidal efficacy. The killing rate and potency varied between honeys and ranged from over a 6-log10 to 4-log10 CFU/ml reduction of viable cells, equivalent to complete bacterial eradication. The maximal killing was associated with the extensive degradation of bacterial DNA. Honey concentration at which DNA degradation occurred correlated with cell death observed in the concentration-dependent cell-kill on agar plates. There was no quantitative relationship between the ·OH generation by honey and bactericidal effect. At the MBC, where there was no surviving cells and no DNA was visible on agarose gels, the ·OH levels were on average 2–3x lower than at Minimum Inhibitory Concentration (MICs) (p < 0.0001). Pre-treatment of honey with catalase, abolished the bactericidal effect. This raised possibilities that either the abrupt killing prevented accumulation of ·OH (dead cells did not generate ·OH) or that DNA degradation and killing is the actual footprint of ·OH action. In conclusion, honeys of buckwheat origin exhibited powerful, concentration-dependent bactericidal effect. The killing and DNA degradation showed a cause-effect relationship. Hydrogen peroxide was an active part of honey killing mechanism. PMID:22783246

Brudzynski, Katrina; Abubaker, Kamal; Wang, Tony

2012-01-01

147

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

NASA Astrophysics Data System (ADS)

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."

Millard, Julie T.; Pilon, André M.

2003-04-01

148

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

ERIC Educational Resources Information Center

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

Millard, Julie T

2011-01-01

149

Interferon Antagonist NSs of La Crosse Virus Triggers a DNA Damage Response-like Degradation of Transcribing RNA Polymerase II*  

PubMed Central

La Crosse encephalitis virus (LACV) is a mosquito-borne member of the negative-strand RNA virus family Bunyaviridae. We have previously shown that the virulence factor NSs of LACV is an efficient inhibitor of the antiviral type I interferon system. A recombinant virus unable to express NSs (rLACVdelNSs) strongly induced interferon transcription, whereas the corresponding wt virus (rLACV) suppressed it. Here, we show that interferon induction by rLACVdelNSs mainly occurs through the signaling pathway leading from the pattern recognition receptor RIG-I to the transcription factor IRF-3. NSs expressed by rLACV, however, acts downstream of IRF-3 by specifically blocking RNA polymerase II-dependent transcription. Further investigations revealed that NSs induces proteasomal degradation of the mammalian RNA polymerase II subunit RPB1. NSs thereby selectively targets RPB1 molecules of elongating RNA polymerase II complexes, the so-called IIo form. This phenotype has similarities to the cellular DNA damage response, and NSs was indeed found to transactivate the DNA damage response gene pak6. Moreover, NSs expressed by rLACV boosted serine 139 phosphorylation of histone H2A.X, one of the earliest cellular reactions to damaged DNA. However, other DNA damage response markers such as up-regulation and serine 15 phosphorylation of p53 or serine 1524 phosphorylation of BRCA1 were not triggered by LACV infection. Collectively, our data indicate that the strong suppression of interferon induction by LACV NSs is based on a shutdown of RNA polymerase II transcription and that NSs achieves this by exploiting parts of the cellular DNA damage response pathway to degrade IIo-borne RPB1 subunits. PMID:21118815

Verbruggen, Paul; Ruf, Marius; Blakqori, Gjon; Överby, Anna K.; Heidemann, Martin; Eick, Dirk; Weber, Friedemann

2011-01-01

150

Trigger-responsive, fast-degradable poly(?-amino ester)s for enhanced DNA unpackaging and reduced toxicity.  

PubMed

Poly(?-amino ester)s (PBAEs) represent an important class of cationic gene delivery materials which, however, suffer from uncontrolled DNA release due in part to the slow degradation of their polyester backbone. Additionally, PBAEs with high molecular weight (MW) also show considerable toxicities. In this study, we designed and developed PBAEs with trigger-responsive domains built-in polymer backbones that can be rapidly cleaved upon external UV light triggering to promote intracellular DNA release as well as reduce material toxicity. Photo-responsive PBAEs were prepared via polyaddition of (2-nitro-1,3-phenylene)bis(methylene) diacrylate and a bifunctional amine. The nitrobenzene moiety was placed in each repeating unit of the PBAE to allow fast response to external UV irradiation, and thus the ester linkers were cleaved and the polymers were degraded within several minutes upon UV irradiation. Cationic PBAEs with high MWs were able to mediate effective intracellular gene delivery, while upon UV irradiation post-transfection, enhanced DNA unpackaging and reduced material toxicity were observed, which collectively contributed to greatly improved transfection efficiencies in various mammalian cell types tested. This strategy allows precise manipulation of material toxicity and gene release profiles of PBAEs, and thus provides an effective design approach to address critical issues in non-viral gene delivery. PMID:24674461

Deng, Xiaojian; Zheng, Nan; Song, Ziyuan; Yin, Lichen; Cheng, Jianjun

2014-06-01

151

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

PubMed Central

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

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

2012-01-01

152

Mechanism of DNA Chain Growth, IV. Direction of Synthesis of T4 Short DNA Chains as Revealed by Exonucleolytic Degradation  

Microsoft Academic Search

T4 nascent short chains labeled at their growing ends with H3-thymidine and uniformly with C14-thymidine were prepared, separated into complementary strands, and degraded by E. coli exonuclease I in the 3' to 5' direction or by B. subtilis nuclease in the 5' to 3' direction. The kinetics of release of H3 and C14 labels by both enzymes was consistent with

Tuneko Okazaki; Reiji Okazaki

1969-01-01

153

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

SciTech Connect

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.

Govindaswami, M.; Feldhake, D.J.; Loper, J.C. [Univ. of Cincinnati Medical Center, OH (United States)

1994-12-31

154

Amplification of DNA markers from scat samples of the least weasel Mustela nivalis nivalis  

Microsoft Academic Search

To test the feasibility of using field-collected scats as a source of DNA in the study of the least weaselMustela nivalis nivalis Linnaeus, 1766, DNA was extracted from scat samples collected from captive weasels using a modified extraction protocol.\\u000a Using universal primers, the control region of the mitochondrial genome was successfully amplified from scat-extracted DNA.\\u000a This amplification resulted in two

Rongjiang Wang; Jodie N. Painter; Ilkka Hanski

2002-01-01

155

The prevalence of mixed DNA profiles in fingernail samples taken from individuals in the general population  

Microsoft Academic Search

The fingernail hyponychium is an isolated area where biological material may accumulate and can provide a valuable source of evidential material in police investigations. DNA transfer between the victim and suspect frequently occurs during violent crimes and in court there is often reasonable doubt that a mixed DNA profile in a fingernail sample has originated from the assault as the

Olivia Cook; Lindsey Dixon

2007-01-01

156

Genomic DNA extraction protocols for bone samples: The comparison of Qiagen and Zymo Research spin columns  

Microsoft Academic Search

The aim of this study was to develop an extraction protocol for bone samples based on ZR Genomic DNA Tissue MicroPrep kit and perform a quantitative comparison with the existing silica extraction protocol based on Qiagen columns and evaluate the effect of demineralization on the quantity of extracted DNA.

Daniel Vanek; Marcela Silerova; Vladislava Urbanova; Lenka Saskova; Jitka Dubska; Michal Beran

157

Impacts of sampling location within a faeces on DNA quality in two carnivore species  

Microsoft Academic Search

We investigated the influence of sampling location within a faeces on DNA quality by sam- pling from both the outside and inside of 25 brown bear (Ursus arctos) scats and the side and the tip of 30 grey wolf (Canis lupus) scats. The outside of the bear scat and side of the wolf scat had significantly lower nuclear DNA microsatellite

J. L. S TENGLEIN; M. D E B ARBA; D. E. A USBAND; L. P. W AITS

2010-01-01

158

A technique for sampling ancient DNA that minimizes damage to museum specimens  

Microsoft Academic Search

Because of the utility of ancient DNA to conservation genetics (Baker 1994), the number of requests to collect tissue from museum specimens has increased. A drawback of consumptive sampling is that it requires removal and destruction of part of the specimen. Epithelium, hair, dried skeletal muscle, and bone have been used as sources of ancient DNA taken from skulls, postcranial

S. M. Wisely; J. E. Maldonado; R. C. Fleischer

2004-01-01

159

MECHANISM OF DNA CHAIN GROWTH, IV. DIRECTION OF SYNTHESIS OF T4 SHORT DNA CHAINS AS REVEALED BY EXONUCLEOLYTIC DEGRADATION*  

PubMed Central

T4 nascent short chains labeled at their growing ends with H3-thymidine and uniformly with C14-thymidine were prepared, separated into complementary strands, and degraded by E. coli exonuclease I in the 3? to 5? direction or by B. subtilis nuclease in the 5? to 3? direction. The kinetics of release of H3 and C14 labels by both enzymes was consistent with the conclusion that the H3 label is at the 3? end of the nascent short chains of both strands and that the short chains are products of discontinuous synthesis in the 5? to 3? direction along the two template strands. PMID:4989398

Okazaki, Tuneko; Okazaki, Reiji

1969-01-01

160

Sample preparation module for bacterial lysis and isolation of DNA from human urine.  

PubMed

Silica impregnated polymer monolithic columns may provide a simple method for lysing and extracting DNA from bacteria inside of microfluidic chips. Here we use Escherichia coli as a test organism for a point of care thermoplastic microfluidic module designed to take in a urine sample, mix it with lysis buffer, and perform a hybrid chemical/mechanical lysis and solid phase extraction of nucleic acids from the sample. To demonstrate proof-of-concept, we doped human hematuric urine samples with E. coli at concentrations ranging from 10(1)-10(5) colony-forming units/mL (CFU/mL) to simulate patient samples. We then performed on-chip lysis and DNA extraction. The bacterial DNA was amplified using real-time PCR demonstrating lysis and isolation down to 10(1) CFU/mL. Results were comparable to a commercial kit at higher concentrations and performed better at recovering DNA at lower concentrations. PMID:19130239

Kulinski, M Dominika; Mahalanabis, Madhumita; Gillers, Sara; Zhang, Jane Y; Singh, Satish; Klapperich, Catherine M

2009-06-01

161

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

PubMed

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

Overballe-Petersen, Søren; Willerslev, Eske

2014-10-01

162

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

SciTech Connect

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.

Shi Tingming [Institut fuer Umweltmedizinische Forschung an der Heinrich-Heine University Duesseldorf gGmbH, auf'm Hennekamp 50, D-40225 Duesseldorf (Germany); Duffin, Rodger [Institut fuer Umweltmedizinische Forschung an der Heinrich-Heine University Duesseldorf gGmbH, auf'm Hennekamp 50, D-40225 Duesseldorf (Germany); Borm, Paul J.A. [Institut fuer Umweltmedizinische Forschung an der Heinrich-Heine University Duesseldorf gGmbH, auf'm Hennekamp 50, D-40225 Duesseldorf (Germany); Li Hui [Institut fuer Umweltmedizinische Forschung an der Heinrich-Heine University Duesseldorf gGmbH, auf'm Hennekamp 50, D-40225 Duesseldorf (Germany); Weishaupt, Christel [Institut fuer Umweltmedizinische Forschung an der Heinrich-Heine University Duesseldorf gGmbH, auf'm Hennekamp 50, D-40225 Duesseldorf (Germany); Schins, Roel P.F. [Institut fuer Umweltmedizinische Forschung an der Heinrich-Heine University Duesseldorf gGmbH, auf'm Hennekamp 50, D-40225 Duesseldorf (Germany)]. E-mail: roel.schins@uni-duesseldorf.de

2006-05-15

163

Flow cytometric analysis of DNA content in spawning and coastal samples of arctic cisco (Coregonus autumnalis)  

E-print Network

FLOW CYTOMETRIC ANALYSIS OF DNA CONTENT IN SPAWNING AND COASTAL SAMPLES OF ARCTIC CISCQ (COREGONUS AUTUMNALIS) A Thesis by SAMUEL FOURNIER LOCKWOOD Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment... of the requirements for the degree of MASTER OF SCIENCE December 1989 Major Subject: Wildlife and Fisheries Sciences FLOW CYTOMETRIC ANALYSIS OF DNA CONTENT IN SPAWNING AND COASTAL SAMPLES OF ARCTIC CISCO (COREGONUS AUTUMNALIS) A Thesis by SAMUEL FOURNIER...

Lockwood, Samuel Fournier

2012-06-07

164

DNA sampling: a method for probing protein binding at specific loci on bacterial chromosomes.  

PubMed

We describe a protocol, DNA sampling, for the rapid isolation of specific segments of DNA, together with bound proteins, from Escherichia coli K-12. The DNA to be sampled is generated as a discrete fragment within cells by the yeast I-SceI meganuclease, and is purified using FLAG-tagged LacI repressor and beads carrying anti-FLAG antibody. We illustrate the method by investigating the proteins bound to the colicin K gene regulatory region, either before or after induction of the colicin K gene promoter. PMID:19181705

Butala, Matej; Busby, Stephen J W; Lee, David J

2009-04-01

165

Degradation of Myogenic Transcription Factor MyoD by the Ubiquitin Pathway In Vivo and In Vitro: Regulation by Specific DNA Binding  

PubMed Central

MyoD is a tissue-specific transcriptional activator that acts as a master switch for skeletal muscle differentiation. Its activity is induced during the transition from proliferating, nondifferentiated myoblasts to resting, well-differentiated myotubes. Like many other transcriptional regulators, it is a short-lived protein; however, the targeting proteolytic pathway and the underlying regulatory mechanisms involved in the process have remained obscure. It has recently been shown that many short-lived regulatory proteins are degraded by the ubiquitin system. Degradation of a protein by the ubiquitin system proceeds via two distinct and successive steps, conjugation of multiple molecules of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Here we show that MyoD is degraded by the ubiquitin system both in vivo and in vitro. In intact cells, the degradation is inhibited by lactacystin, a specific inhibitor of the 26S proteasome. Inhibition is accompanied by accumulation of high-molecular-mass MyoD-ubiquitin conjugates. In a cell-free system, the proteolytic process requires both ATP and ubiquitin and, like the in vivo process, is preceded by formation of ubiquitin conjugates of the transcription factor. Interestingly, the process is inhibited by the specific DNA sequence to which MyoD binds: conjugation and degradation of a MyoD mutant protein which lacks the DNA-binding domain are not inhibited. The inhibitory effect of the DNA requires the formation of a complex between the DNA and the MyoD protein. Id1, which inhibits the binding of MyoD complexes to DNA, abrogates the effect of DNA on stabilization of the protein. PMID:9742084

Abu Hatoum, Ossama; Gross-Mesilaty, Shlomit; Breitschopf, Kristin; Hoffman, Aviad; Gonen, Hedva; Ciechanover, Aaron; Bengal, Eyal

1998-01-01

166

DNA DNA DNA (d)DNA DNA DNA  

E-print Network

DNA DNA DNA DNA DNA DNA DNA DNA [ 2008] (d)DNA DNA DNA DNA 2 3 DNA DNA DNA DNA DNA DNA DNA (a) (c) (b) (d) #12;DNA DNA DNA DNA DNA DNA DNA DNA (b) DNA [Tanaka et al.2008] DNA DNA DNA DNA DNA DNA DNA #12;iGEM MIT MIT

Hagiya, Masami

167

Genetic characteristics of sperm whales sampled during JAPRNII from 2000 to 2007 as revealed by mitochondrial DNA and microsatellite analyses  

Microsoft Academic Search

We analyzed genetic variations at 15 microsatellite DNA loci and mitochondrial DNA (mtDNA) control region sequences in sample of sperm whales collected during JARPNII from 2000 to 2007 in order to examine the effectiveness of these genetic markers for stock structure study of the species. Analyses of mtDNA and microsatellite markers in the total of 45 sperm whales demonstrated that

Naohisa Kanda; Mutsuo Goto; Luis A. Pastene

168

Development of an Alu-based, Real-Time PCR Method for Quantitation of Human DNA in Forensic Samples  

Microsoft Academic Search

Running header: Human Real-Time PCR Quantitation ABSTRACT: 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. We have previously described a method for quantitation of human DNA

Janice A. Nicklas; Eric Buel

169

Aerobic and anaerobic degradation of tannic acid on water samples from Monjolinho reservoir (São Carlos, SP, Brazil).  

PubMed

In order to describe the transformations of tannic acid during its degradation (under aerobic and anaerobic conditions) incubations were performed. To evaluate the oxygen consumption, the tannic acid was added to 1 L of water sample from Monjolinho's reservoir (22 degrees 00'S and 47 degrees 54'W); these solutions were aerated and the dissolved oxygen was monitored for 16 days, the anaerobic process was avoided. For the anaerobic and aerobic degradation, the dissolved organic carbon and the acid tannic concentrations were estimated on the samples days. The results were fitted to first-order kinetic model, being possible to verify that during the 16 days the oxygen uptake was 3.6 mg.L-1, the deoxygenation rate (kD) of this process was 0.39 day-1. The degradation coefficients were calculated through the decay of the tannic acid and organic carbon concentrations. In the aerobic process, the global decay coefficient (kG) was 0.36 day-1 and in the anaerobic 0.28 day-1. Overall, the obtained degradation coefficients suggest that the bacterioplankton of the Monjolinho's reservoir possess a high capacity of polyphenols degradation. PMID:12659006

Cunha-Santino, M B; Bianchini, Júnior I; Serrano, L E F

2002-11-01

170

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

PubMed

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

Karamon, J

2014-01-01

171

Evaluation of DNA extraction kits for molecular diagnosis of human Blastocystis subtypes from fecal samples.  

PubMed

Blastocystis sp. is now recognized as one of the most common intestinal parasite in human fecal examinations. Recently, PCR-based diagnostic methods of Blastocystis infection using direct DNA extraction from fresh fecal samples with commercially available kits are reported. Several kits have been developed, but little has been done in comparing the detective sensitivity between PCR methods using the commercial kits. In this study, we compared the detective sensitivity among five commercially available kits (MagNA Pure LC DNA Isolation Kit I, Roche; QuickGene SP Kit DNA, FujiFilm; NucleoSpin Plant II, Macherey-Nagel; QIAamp DNA Stool Mini Kit, Qiagen; ZR Fecal DNA Kit, Zymo Research) and fecal culture method. In a preliminary test, the DNA isolated with two kits (FujiFilm and Macherey-Nagel) showed negative PCR, while the other three kits showed positive PCR. Then, DNA from 50 clinical samples that was Blastocystis-positive in the examination of fecal culture method were isolated with the three kits and 1.1 kbp SSU rRNA gene was detected with PCR. The positive rates of the three kits (Roche, Qiagen, and Zymo Research) were 10, 48 and 94%, respectively. The present study indicated that there is different detective sensitivity among the commercial kits, and fecal culture method is superior in detection rate and cost performance than DNA-elution kits for diagnosis of Blastocystis sp. subtypes. PMID:21499752

Yoshikawa, Hisao; Dogruman-Al, Funda; Dogruman-Ai, Funda; Turk, Songul; Kustimur, Semra; Balaban, Neriman; Sultan, Nedim

2011-10-01

172

DNA Biosensor for Rapid Detection of Genotoxic Compounds in Soil Samples  

PubMed Central

An electrochemical DNA-based biosensor is proposed as a fast and easy screening method for the detection of genotoxic compounds in soil samples. The biosensor was assembled by immobilising double stranded Calf thymus DNA on screen-printed electrodes. The interactions between DNA and environmental pollutants can cause variations of the electrochemical proprieties of DNA when they cause a DNA damage. Preliminary studies were performed using benzene, naphthalene and anthracene derivatives as model compounds. The effect of these compounds on the surface-confined DNA was found to be linearly related to their concentration in solution. On the other hand, the objective was to optimise the ultrasonic extraction conditions of these compounds from artificially spiked soil samples. Then, the applicability of such a biosensor was evaluated by analysing soil samples from an Italian region with ecological risk (ACNA of Cengio, SV). DNA biosensor for qualitative analysis of soil presented a good correlation with a semi-quantitative method for aromatic ring systems determination as fixed wavelength fluorescence and interestingly, according results were found also with other bioassays. This kind of biosensors represent a new, easy and fast way of analysis of polluted sites, therefore they can be used as early warnings devices in areas with ecological risk as in situ measurement.

Bagni, Graziana; Hernandez, Silvia; Mascini, Marco; Sturchio, Elena; Boccia, Priscilla; Marconi, Simona

2005-01-01

173

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

PubMed Central

Comprehensive sampling is crucial to DNA barcoding, but it is rarely performed because materials are usually unavailable. In practice, only a few rather than all species of a genus are required to be identified. Thus identification of a given species using a limited sample is of great importance in current application of DNA barcodes. Here, we selected 70 individuals representing 48 species from each major lineage of Solanum, one of the most species-rich genera of seed plants, to explore whether DNA barcodes can provide reliable specific-species discrimination in the context of incomplete sampling. Chloroplast genes ndhF and trnS-trnG and the nuclear gene waxy, the commonly used markers in Solanum phylogeny, were selected as the supplementary barcodes. The tree-building and modified barcode gap methods were employed to assess species resolution. The results showed that four Solanum species of quarantine concern could be successfully identified through the two-step barcoding sampling strategy. In addition, discrepancies between nuclear and cpDNA barcodes in some samples demonstrated the ability to discriminate hybrid species, and highlights the necessity of using barcode regions with different modes of inheritance. We conclude that efficient phylogenetic markers are good candidates as the supplementary barcodes in a given taxonomic group. Critically, we hypothesized that a specific-species could be identified from a phylogenetic framework using incomplete sampling–through this, DNA barcoding will greatly benefit the current fields of its application. PMID:23409092

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

2013-01-01

174

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

PubMed

A detection system for Legionella DNA in urine samples based on the polymerase chain reaction (PCR) was developed and tested on infected guinea pigs and patients suffering from pneumonia. Results were compared with standard methods for diagnosis of Legionnaires' disease. A primer system was selected which amplifies a 108 bp DNA fragment of the 5S rRNA gene. The sensitivity of the PCR system was one femtogram of extracted Legionella DNA. Three methods were tested for pretreatment of urine samples. Of these, the Geneclean II kit (Bio 101, USA) gave the best results for artificially contaminated urine samples as well as those from infected guinea pigs or patients. Thirty-seven urine samples from 15 guinea pigs intraperitoneally infected with either Legionella pneumophila serogroup 1, 3 and 6 or Legionella micdadei, 26 urine samples of 21 patients suffering from pneumonia, and 30 control samples of patients with urinary tract infection (UTI) were tested. Legionella DNA was detected in 29 of the guinea pig urine samples; whereas, urinary antigen detection using EIA was positive in only 20 of the samples. PCR was also positive in the samples of 11 patients with pneumonia, 9 of which were confirmed by other microbiological methods, such as culture, direct fluorescent antibody test, urinary antigen detection and antibody testing. However, of the 30 control samples from patients with UTI, three samples yielded positive results. The results demonstrate that Legionella DNA is excreted in the urine of infected individuals and that the PCR shows a higher degree of sensitivity than EIA to the detection of soluble Legionella antigen in urine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7729449

Maiwald, M; Schill, M; Stockinger, C; Helbig, J H; Lück, P C; Witzleb, W; Sonntag, H G

1995-01-01

175

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

PubMed Central

Our application of transition path sampling to a complex biomolecular system in explicit solvent, the closing transition of DNA polymerase ?, unravels atomic and energetic details of the conformational change that precedes the chemical reaction of nucleotide incorporation. The computed reaction profile offers detailed mechanistic insights into, as well as kinetic information on, the complex process essential for DNA synthesis and repair. The five identified transition states extend available experimental and modeling data by revealing highly cooperative dynamics and critical roles of key residues (Arg-258, Phe-272, Asp-192, and Tyr-271) in the enzyme's function. The collective cascade of these sequential conformational changes brings the DNA/DNA polymerase ? system to a state nearly competent for the chemical reaction and suggests how subtle residue motions and conformational rate-limiting steps affect reaction efficiency and fidelity; this complex system of checks and balances directs the system to the chemical reaction and likely helps the enzyme discriminate the correct from the incorrect incoming nucleotide. Together with the chemical reaction, these conformational features may be central to the dual nature of polymerases, requiring specificity (for correct nucleotide selection) as well as versatility (to accommodate different templates at every step) to maintain overall fidelity. Besides leading to these biological findings, our developed protocols open the door to other applications of transition path sampling to long-time, large-scale biomolecular reactions. PMID:15069184

Radhakrishnan, Ravi; Schlick, Tamar

2004-01-01

176

Novel circular DNA viruses in stool samples of wild-living chimpanzees  

PubMed Central

Viral particles in stool samples from wild-living chimpanzees were analysed using random PCR amplification and sequencing. Sequences encoding proteins distantly related to the replicase protein of single-stranded circular DNA viruses were identified. Inverse PCR was used to amplify and sequence multiple small circular DNA viral genomes. The viral genomes were related in size and genome organization to vertebrate circoviruses and plant geminiviruses but with a different location for the stem–loop structure involved in rolling circle DNA replication. The replicase genes of these viruses were most closely related to those of the much smaller (?1?kb) plant nanovirus circular DNA chromosomes. Because the viruses have characteristics of both animal and plant viruses, we named them chimpanzee stool-associated circular viruses (ChiSCV). Further metagenomic studies of animal samples will greatly increase our knowledge of viral diversity and evolution. PMID:19759238

Blinkova, Olga; Victoria, Joseph; Li, Yingying; Keele, Brandon F.; Sanz, Crickette; Ndjango, Jean-Bosco N.; Peeters, Martine; Travis, Dominic; Lonsdorf, Elizabeth V.; Wilson, Michael L.; Pusey, Anne E.; Hahn, Beatrice H.; Delwart, Eric L.

2010-01-01

177

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

Microsoft Academic Search

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

Carolina Quesada-Molina; Ana M. García-Campaña; Laura del Olmo-Iruela; Monsalud del Olmo

2007-01-01

178

ATM kinase is a master switch for the ?Np63? phosphorylation/degradation in human head and neck squamous cell carcinoma cells upon DNA damage  

PubMed Central

We previously found that the pro-apoptotic DNA damaging agent, cisplatin, mediated the proteasome-dependent degradation of ?Np63? associated with its increased phosphorylated status. Since ?Np63? usually plays an opposite role to p53 and TAp63 in human cancers, we tested the notion that phosphorylation events induced by DNA damage would affect the protein degradation of ?Np63? in HNSCC cells upon cisplatin exposure. We found that ?Np63? is phosphorylated in the time-dependent fashion at the following positions: S385, T397 and S466, which were surrounded by recognition motifs for ATM, CDK2 and p70s6K kinases, respectively. We showed that chemical agents or siRNA inhibiting the activity of ATM, CDK2 and p70s6K kinases blocked degradation of ?Np63? in HNSCC cells after cisplatin exposure. Site-specific mutagenesis of ?Np63? residues targeted for phosphorylation by ATM, CDK2 or p70s6k led to dramatic modulation of ?Np63? degradation. Finally, we demonstrated that the ?Np63? protein is a target for direct in vitro phosphorylation by ATM, CDK2 or p70s6K. Our results implicate specific kinases, and target phosphorylation sites in the degradation of ?Np63? following DNA damage. PMID:18769144

Huang, Yiping; Sen, Tanusree; Nagpal, Jatin; Upadhyay, Sunil; Trink, Barry; Ratovitski, Edward; Sidransky, David

2011-01-01

179

Impacts of sampling location within a faeces on DNA quality in two carnivore species.  

PubMed

We investigated the influence of sampling location within a faeces on DNA quality by sampling from both the outside and inside of 25 brown bear (Ursus arctos) scats and the side and the tip of 30 grey wolf (Canis lupus) scats. The outside of the bear scat and side of the wolf scat had significantly lower nuclear DNA microsatellite allelic dropout error rates (U. arctos: P?=?0.017; C. lupus: P?=?0.025) and significantly higher finalized genotyping success rates (U. arctos: P?=?0.017; C. lupus: P?=?0.012) than the tip and inside of the scat. A review of the faecal DNA literature indicated that <45% of studies report the sampling location within a faeces indicating that this methodological consideration is currently underappreciated. Based on our results, we recommend sampling from the side of canid scats and the outside portion of ursid scats to obtain higher quality DNA samples. The sampling location within a faeces should be carefully considered and reported as it can directly influence laboratory costs and efficiency, as well as the ability to obtain reliable genotypes. PMID:21564995

Stenglein, J L; DE Barba, M; Ausband, D E; Waits, L P

2010-01-01

180

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

PubMed Central

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

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

2014-01-01

181

NMR spectroscopic characterisation of oligosaccharides from two Ulva rigida ulvan samples (Ulvales, Chlorophyta) degraded by a lyase  

Microsoft Academic Search

The chemical structure and the sequence of repeating units in ulvans of similar compositions from two different Ulva rigida samples collected in the Canary Islands and in Brittany were studied after ulvan-lyase degradation and NMR spectroscopic analysis of the reaction products. Both ulvans were composed of ulvanobiuronic acid 3-sulfate type A [?4)-?-d-GlcA-(1?4)-?-l-Rha 3-sulfate-(1?] (symbolised as A3s) and contained disaccharides composed

Marc Lahaye

1998-01-01

182

DNA Recognition Process of the Lactose Repressor Protein Studied via Metadynamics and Umbrella Sampling Simulations.  

PubMed

The lactose repressor, LacI, finds its DNA target sites via a process that is faster than what it is expected from a diffusion-driven mechanism. This is possible thanks to nonspecific binding of LacI to DNA, followed by diffusion along the DNA molecule. The diffusion of the protein along DNA might lead to a fast-searching mechanism only if LacI binds with comparable strength to different nonspecific sequences and if, in addition, the value of the binding energy remarkably decreases in the presence of a binding site. The first condition would be favored by loose interactions with the base edges, while the second would take advantage from the opposite situation. In order to understand how the protein satisfies these two opposing requirements, the DNA recognition process was studied by a combination of umbrella sampling and metadynamics simulations. The simulations revealed that when aligned with a specific sequence, LacI establishes polar interactions with the base edges that require ?4 kcal/mol to be disrupted. In contrast, these interactions are not stable when the protein is aligned with nonspecific sequences. These results confirm that LacI is able to efficiently recognize a specific sequence while sliding along DNA before any structural change of the protein-DNA complex occurs. PMID:25341013

Furini, Simone; Domene, Carmen

2014-11-20

183

Autoschizis: a new form of cell death for human ovarian carcinoma cells following ascorbate:menadione treatment. Nuclear and DNA degradation.  

PubMed

Microscopic aspects, densitometric evaluation of Feulgen-stained DNA, and gel electrophoresis of total DNA have been used to elucidate the effects of 1, 2, and 3 h VC (ascorbic acid), VK3 (menadione), and combined VC:VK3 treatments on the cellular and nuclear morphology and DNA content of a human ovarian carcinoma cell line (MDAH 2774). Optical densitometry showed a significant decrease in cancer cell DNA content directly related to VC and VC:VK3 treatments while VK3 and VC:VK3 treated cells exhibited cytoskeletal changes that included self-excision of cytoplasmic pieces with no membranous organelles. Nuclei decreased in size and exhibited poor contrast consistent with progressive decondensation of their chromatin. Degraded chromatin was also detected in cytoplasmic autophagosomes. Nucleoli segregated their components and fragmented into small pieces. Gel electrophoretic analysis of total DNA revealed evidence of generalized DNA degradation specific to treated tumor cells. These results are consistent with previous observations [Scanning 20 (1998a) 564; Ultrastruct. Pathol. 25 (2001b) 183; J. Histochem. Cytochem. 49 (2001) 109] which demonstrated that the VC:VK3 combination induced autoschizic cell death by a series of cytoplasmic excisions without organelles along with specific nuclear ultrastructural damage. PMID:15140597

Gilloteaux, Jacques; Jamison, James M; Lorimer, Heather E; Jarjoura, David; Taper, Henryk S; Calderon, Pedro B; Neal, Deborah R; Summers, Jack L

2004-06-01

184

Occurrence of [i]Leptospira[/i] DNA in water and soil samples collected in eastern Poland.  

PubMed

Leptospira is an important re-emerging zoonotic human pathogen, disseminated by sick and carrier animals, water and soil. Weather calamities, such as flooding or cyclones favour the spreading of these bacteria. To check a potential role of natural water and soil in the persistence and spread of Leptospira on the territory of eastern Poland, 40 samples of natural water and 40 samples of soil were collected from areas exposed to flooding, and 64 samples of natural water and 68 samples of soil were collected from areas not exposed to flooding. Samples of water were taken from various reservoirs (rivers, natural lakes, artificial lakes, canals, ponds, farm wells) and samples of soils were taken at the distance of 1-3 meters from the edge of the reservoirs. The samples were examined for the presence of Leptospira DNA by nested-PCR. Two out of 40 samples of water (5.0%) collected from the area exposed to flooding showed the presence of Leptospira DNA, while all 40 samples of soil from this area were negative. All samples of water and soil (64 and 68, respectively) collected from the areas not exposed to flooding were negative. No significant difference were found between the results obtained in the areas exposed and not exposed to flooding. In conclusion, these results suggest that water and soil have only limited significance in the persistence and dissemination of Leptospira in eastern Poland. PMID:25528911

Wójcik-Fatla, Angelina; Zaj?c, Violetta; Wasi?ski, Bernard; Sroka, Jacek; Cisak, Ewa; Sawczyn, Anna; Dutkiewicz, Jacek

2014-11-26

185

qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.

Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Gallati, Sabina, E-mail: sabina.gallati@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)] [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Schaller, Andre, E-mail: andre.schaller@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)] [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)

2012-07-06

186

Effective detection of rare variants in pooled DNA samples using Cross-pool tailcurve analysis  

PubMed Central

Sequencing targeted DNA regions in large samples is necessary to discover the full spectrum of rare variants. We report an effective Illumina sequencing strategy utilizing pooled samples with novel quality (Srfim) and filtering (SERVIC4E) algorithms. We sequenced 24 exons in two cohorts of 480 samples each, identifying 47 coding variants, including 30 present once per cohort. Validation by Sanger sequencing revealed an excellent combination of sensitivity and specificity for variant detection in pooled samples of both cohorts as compared to publicly available algorithms. PMID:21955804

2011-01-01

187

Quantification of human mitochondrial DNA using synthesized DNA standards.  

PubMed

Successful mitochondrial DNA (mtDNA) forensic analysis depends on sufficient quantity and quality of mtDNA. A real-time quantitative PCR assay was developed to assess such characteristics in a DNA sample, which utilizes a duplex, synthetic DNA to ensure optimal quality assurance and quality control. The assay's 105-base pair target sequence facilitates amplification of degraded DNA and is minimally homologous to nonhuman mtDNA. The primers and probe hybridize to a region that has relatively few sequence polymorphisms. The assay can also identify the presence of PCR inhibitors and thus indicate the need for sample repurification. The results show that the assay provides information down to 10 copies and provides a dynamic range spanning seven orders of magnitude. Additional experiments demonstrated that as few as 300 mtDNA copies resulted in successful hypervariable region amplification, information that permits sample conservation and optimized downstream PCR testing. The assay described is rapid, reliable, and robust. PMID:21883207

Kavlick, Mark F; Lawrence, Helen S; Merritt, R Travis; Fisher, Constance; Isenberg, Alice; Robertson, James M; Budowle, Bruce

2011-11-01

188

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

SciTech Connect

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

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

1995-12-31

189

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

Microsoft Academic Search

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

D. A. Bass; J. S. Yaeger; J. T. Kiely; J. S. Crain; L. M. Shem; M. J. Gowdy; M. Besmer; G. B. Mohrman

1995-01-01

190

Filtering "genic" open reading frames from genomic DNA samples for advanced annotation  

PubMed Central

Background In order to carry out experimental gene annotation, DNA encoding open reading frames (ORFs) derived from real genes (termed "genic") in the correct frame is required. When genes are correctly assigned, isolation of genic DNA for functional annotation can be carried out by PCR. However, not all genes are correctly assigned, and even when correctly assigned, gene products are often incorrectly folded when expressed in heterologous hosts. This is a problem that can sometimes be overcome by the expression of protein fragments encoding domains, rather than full-length proteins. One possible method to isolate DNA encoding such domains would to "filter" complex DNA (cDNA libraries, genomic and metagenomic DNA) for gene fragments that confer a selectable phenotype relying on correct folding, with all such domains present in a complex DNA sample, termed the “domainome”. Results In this paper we discuss the preparation of diverse genic ORF libraries from randomly fragmented genomic DNA using ß-lactamase to filter out the open reading frames. By cloning DNA fragments between leader sequences and the mature ß-lactamase gene, colonies can be selected for resistance to ampicillin, conferred by correct folding of the lactamase gene. Our experiments demonstrate that the majority of surviving colonies contain genic open reading frames, suggesting that ß-lactamase is acting as a selectable folding reporter. Furthermore, different leaders (Sec, TAT and SRP), normally translocating different protein classes, filter different genic fragment subsets, indicating that their use increases the fraction of the “domainone” that is accessible. Conclusions The availability of ORF libraries, obtained with the filtering method described here, combined with screening methods such as phage display and protein-protein interaction studies, or with protein structure determination projects, can lead to the identification and structural determination of functional genic ORFs. ORF libraries represent, moreover, a useful tool to proceed towards high-throughput functional annotation of newly sequenced genomes. PMID:21810207

2011-01-01

191

Rapid multi sample DNA amplification using rotary-linear polymerase chain reaction device (PCRDisc)  

PubMed Central

Multiple sample DNA amplification was done by using a novel rotary-linear motion polymerase chain reaction (PCR) device. A simple compact disc was used to create the stationary sample chambers which are individually temperature controlled. The PCR was performed by shuttling the samples to different temperature zones by using a combined rotary-linear movement of the disc. The device was successfully used to amplify up to 12 samples in less than 30?min with a sample volume of 5??l. A simple spring loaded heater mechanism was introduced to enable good thermal contact between the samples and the heaters. Each of the heater temperatures are controlled by using a simple proportional–integral–derivative pulse width modulation control system. The results show a good improvement in the amplification rate and duration of the samples. The reagent volume used was reduced to nearly 25% of that used in conventional method. PMID:22685508

Sugumar, D.; Kong, L. X.; Ismail, Asma; Ravichandran, M.; Su Yin, Lee

2012-01-01

192

Antibiotic Resistance Genes in the Bacteriophage DNA Fraction of Environmental Samples  

PubMed Central

Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, ?-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to ?-lactam antibiotics is conferred by ?-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to ?-lactam antibiotics, namely two ?-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment. PMID:21390233

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

2011-01-01

193

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

PubMed Central

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

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

2014-01-01

194

Ternary Monolayers as DNA Recognition Interfaces for Direct and Sensitive Electrochemical Detection in Untreated Clinical Samples  

PubMed Central

Detection of specific DNA sequences in clinical samples is a key goal of studies on DNA biosensors and gene chips. Herein we present a highly sensitive electrochemical genosensor for direct measurements of specific DNA sequences in undiluted and untreated human serum and urine samples. Such genosensing relies on a new ternary interface involving hexanedithiol (HDT) co-immobilized with the thiolated capture probe (SHCP) on gold surfaces, followed by the incorporation of 6-mercapto-1-hexanol (MCH) as diluent. The performance of ternary monolayers prepared with linear dithiols of different lengths was systematically examined, compared and characterized by cyclic voltammetry and electrochemical impedance spectroscopy, with HDT exhibiting the most favorable analytical performance. The new SHCP/HDT+MCH monolayer led to a 80-fold improvement in the signal-to-noise ratio (S/N) for 1 nM target DNA in undiluted human serum over the common SHCP/MCH binary alkanethiol interface, and allowed the direct quantification of the target DNA down to 7 pM (28 amol) and 17 pM (68 amol) in undiluted/untreated serum and urine, respectively. It also displayed attractive antifouling properties, as indicated from the favorable S/N obtained after a prolonged exposure (24 h) to untreated biological matrices. These attractive features of the SHCP/HDT+MCH sensor interface indicate considerable promise for a wide range of clinical applications. PMID:21377347

Campuzano, Susana; Kuralay, Filiz; Lobo-Castañón, M. Jesús; Bartošík, Martin; Vyavahare, Kedar; Pale?ek, Emil; Haake, David A.; Wang, Joseph

2011-01-01

195

Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples.  

PubMed

Forensic scientists are constantly searching for better, faster, and less expensive ways to increase the first-pass success rate of forensic sample analysis. Technological advances continue to increase the sensitivity of analysis methods to enable genotyping of samples containing minimal amounts of DNA, yet few tools are available that can simultaneously alert the analyst to both the presence of inhibition and level of degradation in samples prior to genotyping to allow analysts the opportunity to make appropriate modifications to their protocols and, consequently, to use less sample. Our laboratory developed a multiplex quantitative PCR assay that amplifies two human nuclear DNA target sequences of different length to assess DNA degradation and a third amplification target, a synthetic oligonucleotide internal PCR control (IPC), to allow for the assessment of PCR inhibition. We chose the two nuclear targets to provide quantity and fragment-length information relevant to the STR amplification targets commonly used for forensic genotyping. The long target (nuTH01, 170-190 bp) spans the TH01 STR locus and uses a FAM-labeled TaqMan probe for detection. The short nuclear target (nuCSF, 67 bp) is directed at the upstream flanking region of the CSF1PO STR locus and is detected using a VIC-labeled TaqManMGB probe. The IPC target sequence is detected using a NED-labeled TaqManMGB probe. The assay was validated on the Applied Biosystems 7500 Real-Time PCR system, which is optimized for NED detection. We report the results of a developmental validation in which the assay was rigorously tested, in accordance with the current SWGDAM guidelines, for precision, sensitivity, accuracy, reproducibility, species specificity, and stability. PMID:17071034

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

2007-07-20

196

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

PubMed

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

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

2012-07-01

197

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

Microsoft Academic Search

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

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

198

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

NASA Technical Reports Server (NTRS)

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

1978-01-01

199

CHEMICAL CHARACTERIZATION OF POLYNUCLEAR AROMATIC HYDROCARBON DEGRADATION PRODUCTS FROM SAMPLING ARTIFACTS  

EPA Science Inventory

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

200

Evaluation of Sample Stability and Automated DNA Extraction for Fetal Sex Determination Using Cell-Free Fetal DNA in Maternal Plasma  

PubMed Central

Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. Methods. A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. Results. Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35?GE/mL versus 259,43?GE/mL), the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. Conclusion. We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe. PMID:24222898

Ordoñez, Elena; Rueda, Laura; Cañadas, M. Paz; Fuster, Carme; Cirigliano, Vincenzo

2013-01-01

201

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

PubMed Central

Reliable detection of viral DNA in stored newborn screening cards (NSC) would give important insight into possible silent infection during pregnancy and around birth. We sought a DNA extraction method with sufficient sensitivity to detect low copy numbers of viral DNA from small punch samples of NSC. Blank NSC were spotted with seronegative EDTA-blood and seropositive EBV EDTA-blood. DNA was extracted with commercial and noncommercial DNA extraction methods and quantified on a spectrofluorometer using a PicoGreen dsDNA quantification kit. Serial dilutions of purified viral DNA controls determined the sensitivity of the amplification protocol, and seropositive EBV EDTA-blood amplified by nested PCR (nPCR) validated the DNA extraction methods. There were considerable differences between the commercial and noncommercial DNA extraction methods (P=0.014; P=0.016). Commercial kits compared favorably, but the QIamp DNA micro kit with an added forensic filter step was marginally more sensitive. The mean DNA yield from this method was 3 ng/?l. The limit of detection was 10 viral genome copies in a 50-?l reaction. EBV nPCR detection in neat and 1:10 diluted DNA extracts could be replicated reliably. We conclude that the QIamp Micro DNA extraction method with the added forensic spin-filter step was suitable for retrospective DNA viral assays from NSC. PMID:21455476

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

2011-01-01

202

Development of a real-time PCR to detect Demodex canis DNA in different tissue samples.  

PubMed

The present study reports the development of a real-time polymerase chain reaction (PCR) to detect Demodex canis DNA on different tissue samples. The technique amplifies a 166 bp of D. canis chitin synthase gene (AB 080667) and it has been successfully tested on hairs extracted with their roots and on formalin-fixed paraffin embedded skin biopsies. The real-time PCR amplified on the hairs of all 14 dogs with a firm diagnosis of demodicosis and consistently failed to amplify on negative controls. Eleven of 12 skin biopsies with a morphologic diagnosis of canine demodicosis were also positive. Sampling hairs on two skin points (lateral face and interdigital skin), D. canis DNA was detected on nine of 51 healthy dogs (17.6%) a much higher percentage than previously reported with microscopic studies. Furthermore, it is foreseen that if the number of samples were increased, the percentage of positive dogs would probably also grow. Moreover, in four of the six dogs with demodicosis, the samples taken from non-lesioned skin were positive. This finding, if confirmed in further studies, suggests that demodicosis is a generalized phenomenon in canine skin, due to proliferation of local mite populations, even though macroscopic lesions only appear in certain areas. The real-time PCR technique to detect D. canis DNA described in this work is a useful tool to advance our understanding of canine demodicosis. PMID:20865428

Ravera, Ivan; Altet, Laura; Francino, Olga; Bardagí, Mar; Sánchez, Armand; Ferrer, Lluís

2011-02-01

203

Diagnosis of Schistosoma haematobium by Detection of Specific DNA Fragments from Filtered Urine Samples  

PubMed Central

Definitive diagnosis of Schistosoma haematobium infection in adult patients is a clinically important challenge. Chronically infected adults pass few eggs in the urine, which are often missed when current diagnostic methods are used. In the work presented here, we report on an alternative diagnostic method based on presence of the S. haematobium-specific Dra 1, 121 bp repeat fragment in human urine. A novel method of collecting the urine specimens in the field and filtering them through heavy Whatman No. 3 paper was introduced. After drying, the samples remained viable for several months at room temperature. To test the potential use of this method, 89 urine specimens from school children in Kollo District, Niger, were examined. In all, 52 of 89 (58.4%) were positive for hematuria, 4 of 89 (49.4%) were positive for eggs, and 51 of 89 (57.3%) showed parasite-specific DNA. These were compared with 60 filtered urine specimens obtained from random samples of adults from two study sites in Nigeria, one endemic and one non-endemic for S. haematobium. In the 30 patients from the endemic site, all 10 samples with detectable eggs and 7 of the 20 egg-negative samples were DNA positive. It was concluded that the urine filter paper method was sufficiently sensitive to detect low and cryptic infections, that DNA detection was more sensitive than egg detection, and that the filtration method facilitated specimen collection and transport from the field. PMID:21633040

Ibironke, Olufunmilola A.; Phillips, Anna E.; Garba, Amadou; Lamine, Sani M.; Shiff, Clive

2011-01-01

204

Diagnosis of Schistosoma haematobium by detection of specific DNA fragments from filtered urine samples.  

PubMed

Definitive diagnosis of Schistosoma haematobium infection in adult patients is a clinically important challenge. Chronically infected adults pass few eggs in the urine, which are often missed when current diagnostic methods are used. In the work presented here, we report on an alternative diagnostic method based on presence of the S. haematobium-specific Dra 1, 121 bp repeat fragment in human urine. A novel method of collecting the urine specimens in the field and filtering them through heavy Whatman No. 3 paper was introduced. After drying, the samples remained viable for several months at room temperature. To test the potential use of this method, 89 urine specimens from school children in Kollo District, Niger, were examined. In all, 52 of 89 (58.4%) were positive for hematuria, 4 of 89 (49.4%) were positive for eggs, and 51 of 89 (57.3%) showed parasite-specific DNA. These were compared with 60 filtered urine specimens obtained from random samples of adults from two study sites in Nigeria, one endemic and one non-endemic for S. haematobium. In the 30 patients from the endemic site, all 10 samples with detectable eggs and 7 of the 20 egg-negative samples were DNA positive. It was concluded that the urine filter paper method was sufficiently sensitive to detect low and cryptic infections, that DNA detection was more sensitive than egg detection, and that the filtration method facilitated specimen collection and transport from the field. PMID:21633040

Ibironke, Olufunmilola A; Phillips, Anna E; Garba, Amadou; Lamine, Sani M; Shiff, Clive

2011-06-01

205

Effect of DNA Extraction Methods and Sampling Techniques on the Apparent Structure of Cow and Sheep Rumen Microbial Communities  

PubMed Central

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 from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided. PMID:24040342

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

2013-01-01

206

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

PubMed

A competitive enzyme-linked immunosorbent assay (ELISA), the most frequently used immunoassay for the determination of polycyclic aromatic hydrocarbon-DNA adducts in human tissues, has been modified to achieve approximately a 6-fold increase in sensitivity. The new assay, a competitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) has utilized the same rabbit antiserum as the ELISA, antiserum elicited against DNA modified with benzo[a]pyrene. However, the alkaline phosphatase conjugate has been replaced with a biotin-europium-labeled streptavidin signal amplification system, and the release of europium into the solution forms a highly fluorescent chelate complex that is measured by time-resolved fluorometry. The DELFIA has achieved a 5- to 6-fold increase in sensitivity for measurement of DNA samples modified in vitro with benzo[a]pyrene, for cultured cells exposed to radiolabeled benzo[a]pyrene, and for human samples from occupationally exposed workers. The assay has been validated by comparison of adduct levels determined by DELFIA, ELISA, and radioactivity in DNA from mouse keratinocytes exposed to radiolabeled benzo[a]pyrene. Human lymphocyte DNA samples from 104 Hungarian aluminum plant workers were assayed by ELISA and compared to blood cell DNA samples from 69 Italian coke oven workers assayed by DELFIA. The standard curves demonstrated that the limit of detection of 4.0 adducts in 10(8) nucleotides for polycyclic aromatic hydrocarbon-DNA adducts by ELISA, using 35 micrograms of DNA/microtiter plate well, has been decreased to 1.3 adducts in 10(8) nucleotides by DELFIA, using 20 micrograms of DNA/microtiter well. If 35 micrograms of DNA were used in the DELFIA, the calculated detection limit would be 0.7 adducts in 10(8) nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8348058

Schoket, B; Doty, W A; Vincze, I; Strickland, P T; Ferri, G M; Assennato, G; Poirier, M C

1993-01-01

207

Synthesis, characterization, molecular modeling and eukaryotic DNA degradation of 1-(3,4-dihydroxybenzylidene)thiosemicarbazide complexes  

NASA Astrophysics Data System (ADS)

A new chelating agent, 1-(3,4-dihydroxybenzylidene)thiosemicarbazide, H 3BTS, has been introduced for complexation with AsO 2+, SbO +, VO 2+, Cr(III), Fe(III), Co(II), Ni(II), Cu(II), Zn(II) and Bi(III) ions. The isolated chelates are characterized by partial elemental analyses, magnetic moments, spectra (IR, UV-Vis, ESR) and thermal studies. The molecular parameters of the ligand and its metal complexes have been calculated. The protonation constants of H 3BTS (10.35, 9.45 and 8.35) and the stepwise stability constants of its complexes are calculated; Fe(III)-H 3BTS system was found the most stable while Cr(III)-H 3BTS was the lowest. The ligand coordinates as tribasic with Fe(III), Ni(II) and SbO +, dibasic with VO 2+, Cu(II), Co(II), Bi(III), AsO 2+ and Cr(III) and monobasic with Zn(II) ions. Copper(II) and chromium(III) complexes measured anomalous magnetic moments while Co(II), Ni(II) and Fe(III) complexes have normal values. The ligand field parameters were calculated for [Cr 2(HL)(OAc) 2(OH) 2(H 2O)] and [Co(HL)(H 2O) 2] and their values were found in the range reported for a tetrahedral structure. The ESR spectra of the Cu(II) and VO 2+ complexes support the binuclear structure. The end product in the thermal decomposition of some complexes are the metals (Bi, As and/or Sb). [(VO) 2(HL)(SO 4)]2H 2O, [Cr 2(HL)(OAc) 2(OH) 2(H 2O)] and [Co(HL)(H 2O) 2] are found active for the degradation of the DNA of eukaryotic subject completely.

El-Asmy, Ahmed A.; Al-Gammal, Ola A.; Saad, Dena A.; Ghazy, Shaban E.

2009-09-01

208

Spectral, thermal, kinetic, molecular modeling and eukaryotic DNA degradation studies for a new series of albendazole (HABZ) complexes.  

PubMed

This work represents the elaborated investigation for the ligational behavior of the albendazole ligand through its coordination with, Cu(II), Mn(II), Ni(II), Co(II) and Cr(III) ions. Elemental analysis, molar conductance, magnetic moment, spectral studies (IR, UV-Vis and ESR) and thermogravimetric analysis (TG and DTG) have been used to characterize the isolated complexes. A deliberate comparison for the IR spectra reveals that the ligand coordinated with all mentioned metal ions by the same manner as a neutral bidentate through carbonyl of ester moiety and NH groups. The proposed chelation form for such complexes is expected through out the preparation conditions in a relatively acidic medium. The powder XRD study reflects the amorphous nature for the investigated complexes except Mn(II). The conductivity measurements reflect the non-electrolytic feature for all complexes. In comparing with the constants for the magnetic measurements as well as the electronic spectral data, the octahedral structure was proposed strongly for Cr(III) and Ni(II), the tetrahedral for Co(II) and Mn(II) complexes but the square-pyramidal for the Cu(II) one. The thermogravimetric analysis confirms the presence or absence of water molecules by any type of attachments. Also, the kinetic parameters are estimated from DTG and TG curves. ESR spectrum data for Cu(II) solid complex confirms the square-pyramidal state is the most fitted one for the coordinated structure. The albendazole ligand and its complexes are biologically investigated against two bacteria as well as their effective effect on degradation of calf thymus DNA. PMID:20934909

El-Metwaly, Nashwa M; Refat, Moamen S

2011-01-01

209

An analysis of the success rate of 908 trace DNA samples submitted to the Crime Sample Database Unit in New Zealand  

Microsoft Academic Search

The Crime Sample Database Unit, part of the Forensic Biology group at the Institute of Environmental Science and Research Limited, receives samples predominantly from unsolved volume crime cases, such as burglaries and car theft, for inclusion on the New Zealand National DNA Databank. A high proportion of these samples are analysed for the presence of low or trace amounts of

SallyAnn Harbison; Marita Fallow; Donna Bushell

2008-01-01

210

Development of a real-time PCR to detect Demodex canis DNA in different tissue samples  

Microsoft Academic Search

The present study reports the development of a real-time polymerase chain reaction (PCR) to detect Demodex canis DNA on different tissue samples. The technique amplifies a 166 bp of D. canis chitin synthase gene (AB 080667) and it has been successfully tested on hairs extracted with their roots and on formalin-fixed\\u000a paraffin embedded skin biopsies. The real-time PCR amplified on the

Ivan Ravera; Laura Altet; Olga Francino; Mar Bardagí; Armand Sánchez; Lluís Ferrer

2011-01-01

211

Use of Buccal Swabs for Sampling DNA from Nestling and Adult Birds  

Microsoft Academic Search

We evaluated the feasibility and efficiency of using swabs to collect buccal epithelial cells from small (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 from 6 4-day-old nestling boreal chickadees (P. hudsonica).

COLLEEN M. HANDEL; LISA M. PAJOT; SANDRA L. TALBOT; GEORGE K. SAGE

2006-01-01

212

Assessment of microbial diversity in human colonic samples by 16S rDNA sequence analysis  

Microsoft Academic Search

The bacterial species diversity of three colonic tissue samples from elderly people was investigated by sequence analysis of randomly cloned eubacterial 16S rDNA. The majority of sequences (87%) clustered within three bacterial groups: (1) Bacteroides; (2) low G+C content Gram-positives related to Clostridium coccoides (cluster XIVa); (3) Gram-positives related to Clostridium leptum (cluster IV). These groups have been shown to

Georgina L Hold; Susan E Pryde; Valerie J Russell; Elizabeth Furrie; Harry J Flint

2002-01-01

213

Evaluation of DNA extraction kits for molecular diagnosis of human Blastocystis subtypes from fecal samples  

Microsoft Academic Search

Blastocystis sp. is now recognized as one of the most common intestinal parasite in human fecal examinations. Recently, PCR-based diagnostic\\u000a methods of Blastocystis infection using direct DNA extraction from fresh fecal samples with commercially available kits are reported. Several kits\\u000a have been developed, but little has been done in comparing the detective sensitivity between PCR methods using the commercial\\u000a kits.

Hisao Yoshikawa; Funda Dogruman-AI; Songul Turk; Semra Kustimur; Neriman Balaban; Nedim Sultan

214

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

PubMed Central

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

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

2013-01-01

215

Stereoselective quantitation of haloxyfop in environment samples and enantioselective degradation in soils.  

PubMed

The chiral separation of haloxyfop enantiomers was first performed on (R, R) Whelk-O1 chiral column (pirkle type) by high-performance liquid chromatography (HPLC). Chromatographic conditions such as mobile phase composition and column temperature were optimized, and the best resolution was obtained using hexane/n-propanol (98/2) with Rs value of 3.43. Chiral residue analysis methods for haloxyfop enantiomers in environmental matrices, such as soil and water, were developed with recoveries ranging from 85.95% to 104.25%. The results showed that these methods were effective enough for detecting the residual enantiomers environmental matrices. The behavior of haloxyfop in four soils was studied and the enantioselective degradation was found with enantiomer fraction values ranging from 0.058 to 0.61. The research work was extremely useful for investigating the fate of individual enantiomers in environment, the mechanism of the stereoselective behaviors, and the risk assessment of chiral pesticide. PMID:25128890

Sun, Mingjing; Liu, Donghui; Shen, Zhigang; Zhou, Zhiqiang; Wang, Peng

2015-01-01

216

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

PubMed

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

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

2013-05-01

217

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...

218

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

PubMed

Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination. PMID:22310206

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

2012-09-01

219

Amplification volume reduction on DNA database samples using FTA™ Classic Cards.  

PubMed

The DNA forensic community always strives towards improvements in aspects such as sensitivity, robustness, and efficacy balanced with cost efficiency. Therefore our laboratory decided to study the feasibility of PCR amplification volume reduction using DNA entrapped in FTA™ Classic Card and to bring cost savings to the laboratory. There were a few concerns the laboratory needed to address. First, the kinetics of the amplification reaction could be significantly altered. Second, an increase in sensitivity might affect interpretation due to increased stochastic effects even though they were pristine samples. Third, statics might cause FTA punches to jump out of its allocated well into another thus causing sample-to-sample contamination. Fourth, the size of the punches might be too small for visual inspection. Last, there would be a limit to the extent of volume reduction due to evaporation and the possible need of re-injection of samples for capillary electrophoresis. The laboratory had successfully optimized a reduced amplification volume of 10 ?L for FTA samples. PMID:21543276

Wong, Hang Yee; Lim, Eng Seng Simon; Tan-Siew, Wai Fun

2012-03-01

220

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

PubMed

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. PMID:21430321

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

221

Demonstration of Coccidioides immitis and Coccidioides posadasii DNA in soil samples collected from Dinosaur National Monument, Utah.  

PubMed

Soil samples were collected in 2006 from Dinosaur National Monument (DNM), Utah, the site of an outbreak of coccidioidomycosis in 2001. DNA was isolated from two soil samples, and polymerase chain reaction (PCR) amplified Coccidioides DNA present in both samples. Ribosomal RNA genes and internal transcribed spacer (ITS) region PCR products were sequenced. Single-nucleotide polymorphisms indicated that the DNA from sample SS06RH was that of Coccidioides immitis, while the DNA from sample SS06UM was C. posadasii. This is the first report to directly demonstrate Coccidioides in soils from DNM and the first to report the presence of both C. immitis and C. posadasii in the same geographic location. PMID:24847036

Johnson, Suzanne M; Carlson, Erin L; Fisher, Frederick S; Pappagianis, Demosthenes

2014-08-01

222

Protocols for metagenomic DNA extraction and Illumina amplicon library preparation for faecal and swab samples.  

PubMed

Next-generation sequencing (NGS) technology has extraordinarily enhanced the scope of research in the life sciences. To broaden the application of NGS to systems that were previously difficult to study, we present protocols for processing faecal and swab samples into amplicon libraries amenable to Illumina sequencing. We developed and tested a novel metagenomic DNA extraction approach using solid phase reversible immobilization (SPRI) beads on Western Bluebird (Sialia mexicana) samples stored in RNAlater. Compared with the MO BIO PowerSoil Kit, the current standard for the Human and Earth Microbiome Projects, the SPRI-based method produced comparable 16S rRNA gene PCR amplification from faecal extractions but significantly greater DNA quality, quantity and PCR success for both cloacal and oral swab samples. We furthermore modified published protocols for preparing highly multiplexed Illumina libraries with minimal sample loss and without post-adapter ligation amplification. Our library preparation protocol was successfully validated on three sets of heterogeneous amplicons (16S rRNA gene amplicons from SPRI and PowerSoil extractions as well as control arthropod COI gene amplicons) that were sequenced across three independent, 250-bp, paired-end runs on Illumina's MiSeq platform. Sequence analyses revealed largely equivalent results from the SPRI and PowerSoil extractions. Our comprehensive strategies focus on maximizing efficiency and minimizing costs. In addition to increasing the feasibility of using minimally invasive sampling and NGS capabilities in avian research, our methods are notably not avian-specific and thus applicable to many research programmes that involve DNA extraction and amplicon sequencing. PMID:24774752

Vo, A-T E; Jedlicka, J A

2014-11-01

223

BSCTV C2 Attenuates the Degradation of SAMDC1 to Suppress DNA Methylation-Mediated Gene Silencing in Arabidopsis[W][OA  

PubMed Central

Plant viruses are excellent tools for studying microbial–plant interactions as well as the complexities of host activities. Our study focuses on the role of C2 encoded by Beet severe curly top virus (BSCTV) in the virus–plant interaction. Using BSCTV C2 as bait in a yeast two-hybrid screen, a C2-interacting protein, S-adenosyl-methionine decarboxylase 1 (SAMDC1), was identified from an Arabidopsis thaliana cDNA library. The interaction was confirmed by an in vitro pull-down assay and a firefly luciferase complemention imaging assay in planta. Biochemical analysis further showed that the degradation of the SAMDC1 protein was inhibited by MG132, a 26S proteasome inhibitor, and that C2 could attenuate the degradation of the SAMDC1 protein. Genetic analysis showed that loss of function of SAMDC1 resulted in reduced susceptibility to BSCTV infection and reduced viral DNA accumulation, similar to the effect of BSCTV C2 deficiency. Bisulfite sequencing analysis further showed that C2 deficiency caused enhanced DNA methylation of the viral genome in infected plants. We also showed that C2 can suppress de novo methylation in the FWA transgenic assay in the C2 transgene background. Overexpression of SAMDC1 can mimic the suppressive activity of C2 against green fluorescent protein–directed silencing. These results suggest that C2 interferes with the host defense mechanism of DNA methylation-mediated gene silencing by attenuating the 26S proteasome-mediated degradation of SAMDC1. PMID:21245466

Zhang, Zhonghui; Chen, Hao; Huang, Xiahe; Xia, Ran; Zhao, Qingzhen; Lai, Jianbin; Teng, Kunling; Li, Yin; Liang, Liming; Du, Quansheng; Zhou, Xueping; Guo, Huishan; Xie, Qi

2011-01-01

224

Degradation of alpha-pinene on Tenax during sample storage: effects of daylight radiation and temperature.  

PubMed

The behavior of alpha-pinene sampled on adsorption cartridges filled with Tenax TA has been investigated in relation to different storage conditions, focusing on daylight radiation and temperature. After sampling, the respective cartridges containing the terpene were placed in sunlight on the windowsill for up to 1 month. Corresponding samples have been wrapped in aluminum foil to prevent the influence of daylight radiation. Additional sample cartridges with alpha-pinene were stored in the refrigerator at 4 degrees C and a freezer at -18 degrees C. All cartridges were analyzed using thermodesorption injection onto a gas chromatograph, and the compounds were detected using either a cryocondensation-interface to a Fourier transform infrared-spectrometer (GC/FT-IR) or the flame ionization detector (FID). In summary, 12 compounds were detected and identified, from which eight were products that were formed on Tenax through different mechanisms. Two compounds seemed to be formed under the influence of daylight radiation, while the others appear to be mainly autoxidation products. Estimates after 1 month of storage showed recoveries of over 99% for wrapped samples, while for unwrapped cartridges only about 88% of alpha-pinene was found. A pattern of up to five compounds was found that can be used as an indicator for storage reactions. PMID:11452597

Schrader, W; Geiger, J; Klockow, D; Korte, E H

2001-07-01

225

New Detection Modality for Label-Free Quantification of DNA in Biological Samples via Superparamagnetic Bead Aggregation  

PubMed Central

Combining DNA and superparamagnetic beads in a rotating magnetic field produces multiparticle aggregates that are visually striking, and enables label-free optical detection and quantification of DNA at levels in the picogram per microliter range. DNA in biological samples can be quantified directly by simple analysis of optical images of microfluidic wells placed on a magnetic stirrer without DNA purification. Aggregation results from DNA/bead interactions driven either by the presence of a chaotrope (a nonspecific trigger for aggregation) or by hybridization with oligonucleotides on functionalized beads (sequence-specific). This paper demonstrates quantification of DNA with sensitivity comparable to that of the best currently available fluorometric assays. The robustness and sensitivity of the method enable a wide range of applications, illustrated here by counting eukaryotic cells. Using widely available and inexpensive benchtop hardware, the approach provides a highly accessible low-tech microscale alternative to more expensive DNA detection and cell counting techniques. PMID:22423674

Leslie, Daniel C.; Li, Jingyi; Strachan, Briony C.; Begley, Matthew R.; Finkler, David; Bazydlo, Lindsay L.; Barker, N. Scott; Haverstick, Doris; Utz, Marcel; Landers, James P.

2012-01-01

226

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

PubMed Central

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

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

2014-01-01

227

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

PubMed

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

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

2014-01-01

228

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

PubMed

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

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

2014-01-01

229

The influence of sample preparation and incubation sequence on in situ dry matter degradability of fresh  

E-print Network

from mono-filamentous polyester bags (43 Nm pore size). The experiment was designed as a factorial plus time per sequence. Preparation methods were fresh or frozen grass which were subsequently oven dried at 60°C or 100°C, microwave dried or not dried. A grass sample was freeze dried to provide the control

Paris-Sud XI, Université de

230

A noninvasive hair sampling technique to obtain high quality DNA from elusive small mammals.  

PubMed

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations. This approach has proven to be especially useful when dealing with rare or elusive species. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas' habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms. PMID:21445038

Henry, Philippe; Henry, Alison; Russello, Michael A

2011-01-01

231

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

SciTech Connect

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

Tan, H.

1999-03-31

232

OLED-based DNA biochip for Campylobacter spp. detection in poultry meat samples.  

PubMed

Integrated biochips are the ideal solution for producing portable diagnostic systems that uncouple diagnosis from centralized laboratories. These portable devices exploit a multi-disciplinary approach, are cost effective and have several advantages including broader accessibility, high sensitivity, quick test results and ease of use. The application of such a device in food safety is considered in this paper. Fluorescence detection of a specific biological probe excited by an optical source is one of the most commonly used methods for quantitative analysis on biochips. In this study, we designed and characterized a miniaturized, highly-sensitive DNA biochip based on a deep-blue organic light-emitting diode. The molecular design of the diode was optimized to excite a fluorophore-conjugated DNA probe and tested using real meat samples to obtain a high sensitivity and specificity against one of the most common poultry meat contaminants: Campylobacter spp. Real samples were analyzed also by classical plate methods and molecular methods to validate the results obtained by the new DNA-biochip. The high sensitivity obtained by the OLED based biochip (0.37ng/?l) and the short time required for the results (about 24h) indicate the usefulness of the system. PMID:25437363

Manzano, Marisa; Cecchini, Francesca; Fontanot, Marco; Iacumin, Lucilla; Comi, Giuseppe; Melpignano, Patrizia

2015-04-15

233

Reduction of DNA contamination in RNA samples for reverse transcription-polymerase chain reaction using selective precipitation by compaction agents.  

E-print Network

Reduction of DNA contamination in RNA samples for reverse transcription-polymerase chain reaction of Chemical and Biomolecular Engineering, 4800 Calhoun Road, S222 Engineering Building 1, University by reverse transcription-polymerase chain reaction (RT-PCR) is DNA contamination, which can produce

Fox, George

234

Analysis and optimization of bulk DNA sampling with binary scoring for germplasm characterization.  

PubMed

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

Reyes-Valdés, M Humberto; Santacruz-Varela, Amalio; Martínez, Octavio; Simpson, June; Hayano-Kanashiro, Corina; Cortés-Romero, Celso

2013-01-01

235

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

Microsoft Academic Search

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

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

2004-01-01

236

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

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

Psifidi, Androniki; Dovas, Chrysostomos I.; Bramis, Georgios; Lazou, Thomai; Russel, Claire L.; Arsenos, Georgios; Banos, Georgios

2015-01-01

237

A novel methyl-binding domain protein enrichment method for identifying genome-wide tissue-specific DNA methylation from nanogram DNA samples  

PubMed Central

Background Growing evidence suggests that DNA methylation plays a role in tissue-specific differentiation. Current approaches to methylome analysis using enrichment with the methyl-binding domain protein (MBD) are restricted to large (?1 ?g) DNA samples, limiting the analysis of small tissue samples. Here we present a technique that enables characterization of genome-wide tissue-specific methylation patterns from nanogram quantities of DNA. Results We have developed a methodology utilizing MBD2b/MBD3L1 enrichment for methylated DNA, kinase pre-treated ligation-mediated PCR amplification (MeKL) and hybridization to the comprehensive high-throughput array for relative methylation (CHARM) customized tiling arrays, which we termed MeKL-chip. Kinase modification in combination with the addition of PEG has increased ligation-mediated PCR amplification over 20-fold, enabling >400-fold amplification of starting DNA. We have shown that MeKL-chip can be applied to as little as 20 ng of DNA, enabling comprehensive analysis of small DNA samples. Applying MeKL-chip to the mouse retina (a limited tissue source) and brain, 2,498 tissue-specific differentially methylated regions (T-DMRs) were characterized. The top five T-DMRs (Rgs20, Hes2, Nfic, Cckbr and Six3os1) were validated by pyrosequencing. Conclusions MeKL-chip enables genome-wide methylation analysis of nanogram quantities of DNA with a wide range of observed-to-expected CpG ratios due to the binding properties of the MBD2b/MBD3L1 protein complex. This methodology enabled the first analysis of genome-wide methylation in the mouse retina, characterizing novel T-DMRs. PMID:23759032

2013-01-01

238

ER stress suppresses DNA double-strand break repair and sensitizes tumor cells to ionizing radiation by stimulating proteasomal degradation of Rad51.  

PubMed

In this study, we provide evidence that endoplasmic reticulum (ER) stress suppresses DNA double-strand break (DSB) repair and increases radiosensitivity of tumor cells by altering Rad51 levels. We show that the ER stress inducer tunicamycin stimulates selective degradation of Rad51 via the 26S proteasome, impairing DSB repair and enhancing radiosensitivity in human lung cancer A549 cells. We also found that glucose deprivation, which is a physiological inducer of ER stress, triggered similar events. These findings suggest that ER stress caused by the intratumoral environment influences tumor radiosensitivity, and that it has potential as a novel target to improve cancer radiotherapy. PMID:24021650

Yamamori, Tohru; Meike, Shunsuke; Nagane, Masaki; Yasui, Hironobu; Inanami, Osamu

2013-10-11

239

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

PubMed Central

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

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

2014-01-01

240

Novel sphingosine-containing analogues selectively inhibit sphingosine kinase (SK) isozymes, induce SK1 proteasomal degradation and reduce DNA synthesis in human pulmonary arterial smooth muscle cells  

PubMed Central

Sphingosine 1-phosphate (S1P) is involved in hyper-proliferative diseases such as cancer and pulmonary arterial hypertension. We have synthesized inhibitors that are selective for the two isoforms of sphingosine kinase (SK1 and SK2) that catalyze the synthesis of S1P. A thiourea adduct of sphinganine (F02) is selective for SK2 whereas the 1-deoxysphinganines 55-21 and 77-7 are selective for SK1. (2S,3R)-1-Deoxysphinganine (55-21) induced the proteasomal degradation of SK1 in human pulmonary arterial smooth muscle cells and inhibited DNA synthesis, while the more potent SK1 inhibitors PF-543 and VPC96091 failed to inhibit DNA synthesis. These findings indicate that moderate potency inhibitors such as 55-21 are likely to have utility in unraveling the functions of SK1 in inflammatory and hyperproliferative disorders. PMID:24396570

Byun, Hoe-Sup; Pyne, Susan; MacRitchie, Neil; Pyne, Nigel J.

2013-01-01

241

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

PubMed Central

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

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

242

Analysis of artificially degraded DNA using STRs and SNPs—results of a collaborative European (EDNAP) exercise  

Microsoft Academic Search

Recently, there has been much debate about what kinds of genetic markers should be implemented as new core loci that constitute national DNA databases. The choices lie between conventional STRs, ranging in size from 100 to 450bp; mini-STRs, with amplicon sizes less than 200bp; and single nucleotide polymorphisms (SNPs). There is general agreement by the European DNA Profiling Group (EDNAP)

L. A. Dixon; A. E. Dobbins; H. K. Pulker; J. M. Butler; P. M. Vallone; M. D. Coble; W. Parson; B. Berger; P. Grubwieser; H. S. Mogensen; N. Morling; K. Nielsen; J. J. Sanchez; E. Petkovski; A. Carracedo; P. Sanchez-Diz; E. Ramos-Luis; M. Bri?n; J. A. Irwin; R. S. Just; O. Loreille; T. J. Parsons; D. Syndercombe-Court; H. Schmitter; B. Stradmann-Bellinghausen; K. Bender; P. Gill

2006-01-01

243

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

USGS Publications Warehouse

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

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

2013-01-01

244

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

PubMed Central

Objective The aim of this study is to report the experience of noninvasive prenatal DNA testing using massively parallel sequencing in an accredited clinical laboratory. Methods Laboratory information was examined for blood samples received for testing between February and November 2012 for chromosome 21 (Chr21), Chr18, and Chr13. Monosomy X (MX) testing was available from July 2012 for cystic hygroma indication. Outcomes were collected from providers on samples with positive results. Results There were 5974 samples tested, and results were issued within an average of 5.1 business days. Aneuploidy was detected in 284 (4.8%) samples (155 Chr21, 66 Chr18, 19 Chr13, 40 MX, and four double aneuploidy). Follow-ups are available for 245/284 (86%), and 77/284 (27.1%) are confirmed, including one double-aneuploidy case concordant with cytogenetics from maternal malignancy. Fourteen (0.2%) discordant (putative false-positive) results (one Chr21, six Chr18, three Chr13, three MX, and one Chr21/13) have been identified. Five (0.08%) false-negative cases are reported (two trisomy 21, two trisomy 18, and one MX). In 170 (2.8%) cases, the result for a single chromosome was indefinite. Conclusions This report suggests that clinical testing of maternal cell-free DNA for fetal aneuploidy operates within performance parameters established in validation studies. Noninvasive prenatal testing is sensitive to biological contributions from placental and maternal sources. ©2013 Verinata Health, Inc. Prenatal Diagnosis published by John Wiley & Sons, Ltd. PMID:23592485

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

2013-01-01

245

Species identification in mammals from mixed biological samples based on mitochondrial DNA control region length polymorphism.  

PubMed

The ability to identify the species origin of an unknown biological sample is relevant in the fields of human and wildlife forensics. However, the detection of several species mixed in the same sample still remains a challenge. We developed and tested a new approach for mammal DNA identification in mixtures of two or three species, based on the analysis of mitochondrial DNA control region interspecific length polymorphism followed by direct sequencing. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and is not based on a pre-defined panel of species. Amplicons can be separated either on agarose gels or using CE. The advantages and limitations of the assay are discussed under different conditions, such as variable template concentration, amplicon sizes and size difference among the amplicons present in the mixture. For the first time, this protocol provides a simple, reliable and flexible method for simultaneous identification of multiple mammalian species from mixtures, without any prior knowledge of the species involved. PMID:19229844

Pun, Ka-Man; Albrecht, Catherine; Castella, Vincent; Fumagalli, Luca

2009-03-01

246

Developmental toxicity and DNA damage from exposure to parking lot runoff retention pond samples in the Japanese medaka (Oryzias latipes).  

PubMed

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

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

247

Developmental toxicity and DNA damage from exposure to parking lot runoff retention pond samples in the Japanese Medaka (Oryzias latipes)  

PubMed Central

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

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

248

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

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. PMID:25495198

Willison, Stuart A

2015-01-20

249

Development of a Novel Self-Enclosed Sample Preparation Device for DNA/RNA Isolation in Space  

NASA Technical Reports Server (NTRS)

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.

Zhang, Ye; Mehta, Satish K.; Pensinger, Stuart J.; Pickering, Karen D.

2011-01-01

250

NMR spectroscopic characterisation of oligosaccharides from two Ulva rigida ulvan samples (Ulvales, Chlorophyta) degraded by a lyase.  

PubMed

The chemical structure and the sequence of repeating units in ulvans of similar compositions from two different Ulva rigida samples collected in the Canary Islands and in Brittany were studied after ulvan-lyase degradation and NMR spectroscopic analysis of the reaction products. Both ulvans were composed of ulvanobiuronic acid 3-sulfate type A [-->4)-beta-D-GlcA-(1-->4)-alpha-L-Rha 3-sulfate-(1-->] (symbolised as A3s) and contained disaccharides composed of [-->4)-beta-D-Xyl-(1-->4)-alpha-L-Rha 3-sulfate-(1-->] and [-->4)-beta-D-Xyl 2-sulfate-(1-->4)-alpha-L-Rha 3-sulfate], respectively referred to as ulvanobiose 3-sulfate (U3s) and ulvanobiose 2',3-disulfate (U2's,3s). In the Canary Islands sample, these U3s and U2's,3s occurred dispersed among A3s sequences and as short blocks of two or three units. In contrast, in the Brittany samples, these units were dispersed among A3s structures and next to A3s units branched at O-2 of alpha-L-Rha 3-sulfate by a terminal beta-D-GlcA and symbolised as A2g,3s. However, more complex structures are likely to occur in the enzyme resistant fraction remaining from this ulvan. An average structure sequence of these two ulvans was proposed. The transposition of the 13C NMR data of the new identified structures to the parent polysaccharides was not possible, probably due to the different sequence distributions affecting the carbons chemical shifts. PMID:10230036

Lahaye, M

1998-12-21

251

DNA  

NSDL National Science Digital Library

In this activity, students extract DNA from their cheek cells and relate the steps in the procedure to the characteristics of cells and biological molecules. Students learn key concepts about the function of DNA during the intervals required for the extraction procedure. A second optional section develops student understanding of the fundamentals of DNA structure, function and replication; this section includes hands-on modeling of DNA replication. This activity, together with our activity, "From Gene to Protein - Transcription and Translation", can be used to teach the basic concepts of molecular biology.

Doherty, Jennifer; Waldron, Ingrid

252

Reduction of DNA contamination in RNA samples for reverse transcription-polymerase chain reaction using selective precipitation by compaction agents.  

PubMed

An important problem in measurement of messenger RNA (mRNA) levels by reverse transcription-polymerase chain reaction (RT-PCR) is DNA contamination, which can produce artifactually increased mRNA concentration. Current methods to eliminate contaminating DNA can compromise the integrity of the RNA, are time-consuming, and/or are hazardous. We present a rapid, nuclease-free, and cost-effective method of eliminating contaminating DNA in RNA samples using selective precipitation by compaction agents. Compaction agents are cationic molecules that bind to double-stranded nucleic acids, driven by electrostatic interactions and steric complementarity. The effectiveness and DNA selectivity of six compaction agents were investigated: trivalent spermidine, Triquat A, and Triquat 7; tetravalent spermine and Quatro-quat; and hexavalent Quatro-diquat. Effectiveness was measured initially by supernatant UV absorbance after precipitation of salmon sperm DNA. Effectiveness and selectivity were then investigated using differences in RT-PCR C(t) values with synthetic mixtures of human genomic DNA and total RNA and with total RNA isolated from cells. With 500 microM spermidine or Triquat A, the supernatant DNA could not be detected up to 40 cycles of PCR (C(t)12.6), whereas the C(t) for the mRNA was increased by only five cycles. Therefore, spermidine and Triquat A each show strong DNA selectivity and could be used to eliminate contaminating DNA in measurements of mRNA. PMID:18831957

Añez-Lingerfelt, Mariaclara; Fox, George E; Willson, Richard C

2009-01-01

253

DNA.  

ERIC Educational Resources Information Center

Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

Felsenfeld, Gary

1985-01-01

254

Extraction of DNA from plant and fungus tissues in situ  

PubMed Central

Background When samples are collected in the field and transported to the lab, degradation of the nucleic acids contained in the samples is frequently observed. Immediate extraction and precipitation of the nucleic acids reduces degradation to a minimum, thus preserving accurate sequence information. An extraction method to obtain high quality DNA in field studies is described. Findings DNA extracted immediately after sampling was compared to DNA extracted after allowing the sampled tissues to air dry at 21°C for 48 or 72 hours. While DNA extracted from fresh tissues exhibited little degradation, DNA extracted from all tissues exposed to 21°C air for 48 or 72 hours exhibited varying degrees of degradation. Yield was higher for extractions from fresh tissues in most cases. Four microcentrifuges were compared for DNA yield: one standard electric laboratory microcentrifuge (max rcf?=?16,000×g), two battery-operated microcentrifuges (max rcf?=?5,000 and 3,000 ×g), and one manually-operated microcentrifuge (max rcf?=?120×g). Yields for all centrifuges were similar. DNA extracted under simulated field conditions was similar in yield and quality to DNA extracted in the laboratory using the same equipment. Conclusions This CTAB (cetyltrimethylammonium bromide) DNA extraction method employs battery-operated and manually-operated equipment to isolate high quality DNA in the field. The method was tested on plant and fungus tissues, and may be adapted for other types of organisms. The method produced high quality DNA in laboratory tests and under simulated field conditions. The field extraction method should prove useful for working in remote sites, where ice, dry ice, and liquid nitrogen are unavailable; where degradation is likely to occur due to the long distances between the sample site and the laboratory; and in instances where other DNA preservation and transportation methods have been unsuccessful. It may be possible to adapt this method for genomic, metagenomic, transcriptomic and metabolomic projects using samples collected in situ. PMID:22672795

2012-01-01

255

Identification of grass-associated and toluene-degrading diazotrophs, Axoarcus spp., by analyses of partial 16S ribosomal DNA sequences  

SciTech Connect

The genus Azoarcus includes nitrogen-fixing, grass-associated strains as well as denitrifying toluene degraders. In order to identify and group members of the genus Azoarcus, phylogenetic analysis based on partial sequences of 16S rRNA genes (16S rDNAs) is proposed. 16S rRNA-targeted PCR using specific primers to exclude amplification in the majority of other members of the beta subclass of the class Proteobacteria was combined with direct sequencing of the PCR products. Tree inference from comparisons of 446-bp rDNA fragments yielded similar results for the three known Azoarcus spp. sequences and for analysis of the complete 16S rDNA sequence. These three species formed a phylogenetically coherent group with representatives of two other Azoarcus species which were subjected to 16S rRNA sequencing in this study. This group was related to Rhodocyclus purpureus and Thaurea selenatis. New isolates and also sequences of so far uncultured bacteria from roots of Kallar grass were assigned to the genus Azoarcus as well. Also, strains degrading monoaromatic hydrocarbons anaerobically in the presence of nitrate clustered within this genus, albeit not with grass-associated isolates. All representative members of the five species harboring rhizospheric bacteria were able to form N{sub 2}O from nitrate and showed anaerobic growth on malic acid with nitrate but not on toluene. In order to visualize different Azoarcus spp. by whole-cell in situ hybridizations, we generated 16S rRNA-targeted, fluorescent probes by in vitro transcription directly from PCR products which spanned the variable region V2. Hybridization was species specific for Azoarcus communis and Azoarcus indigens. The proposed scheme of phylogenetic analysis of PCR-generated 16S rDNA segements will facilitate studies on ecological distribution, host range, and diversity of Azoarcus spp. and may even allow rapid identification of unc ultured strains from environmental DNAs. 30 refs., 3 figs.

Hurek, T.; Reinhold-Hurek, B. [Max-Planch-Institut fuer Terrestrische Mikrobiologie, Marburg (Germany)

1995-06-01

256

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples  

PubMed Central

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios < 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios > 1.53. However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at ?280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 < ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1). PMID:23407542

Patterson, Jennifer; Mura, Cameron

2013-01-01

257

Insights into biodiversity sampling strategies for freshwater microinvertebrate faunas through bioblitz campaigns and DNA barcoding  

PubMed Central

Background Biodiversity surveys have long depended on traditional methods of taxonomy to inform sampling protocols and to determine when a representative sample of a given species pool of interest has been obtained. Questions remain as to how to design appropriate sampling efforts to accurately estimate total biodiversity. Here we consider the biodiversity of freshwater ostracods (crustacean class Ostracoda) from the region of Churchill, Manitoba, Canada. Through an analysis of observed species richness and complementarity, accumulation curves, and richness estimators, we conduct an a posteriori analysis of five bioblitz-style collection strategies that differed in terms of total duration, number of sites, protocol flexibility to heterogeneous habitats, sorting of specimens for analysis, and primary purpose of collection. We used DNA barcoding to group specimens into molecular operational taxonomic units for comparison. Results Forty-eight provisional species were identified through genetic divergences, up from the 30 species previously known and documented in literature from the Churchill region. We found differential sampling efficiency among the five strategies, with liberal sorting of specimens for molecular analysis, protocol flexibility (and particularly a focus on covering diverse microhabitats), and a taxon-specific focus to collection having strong influences on garnering more accurate species richness estimates. Conclusions Our findings have implications for the successful design of future biodiversity surveys and citizen-science collection projects, which are becoming increasingly popular and have been shown to produce reliable results for a variety of taxa despite relying on largely untrained collectors. We propose that efficiency of biodiversity surveys can be increased by non-experts deliberately selecting diverse microhabitats; by conducting two rounds of molecular analysis, with the numbers of samples processed during round two informed by the singleton prevalence during round one; and by having sub-teams (even if all non-experts) focus on select taxa. Our study also provides new insights into subarctic diversity of freshwater Ostracoda and contributes to the broader “Barcoding Biotas” campaign at Churchill. Finally, we comment on the associated implications and future research directions for community ecology analyses and biodiversity surveys through DNA barcoding, which we show here to be an efficient technique enabling rapid biodiversity quantification in understudied taxa. PMID:23557180

2013-01-01

258

New type of SSUrDNA sequence was detected from both Plasmodium ovale curtisi and Plasmodium ovale wallikeri samples  

PubMed Central

Background Plasmodium ovale is relatively unfamiliar to Chinese staff engaged in malaria diagnosis. In 2013, dried blood spots of four unidentified but suspected ovale malaria samples were sent to the National Malaria Reference Laboratory (NMRL) for reconfirmation. Methods Partial and complete, small, subunit ribosomal DNA (SSU rDNA) sequences of four samples were obtained with PCR-cloning-sequencing method. Obtained sequences were analyzed by aligning with each other and with nine SSU rDNA sequences of six known Plasmodium parasites. A phylogenetic tree was constructed based on complete SSU rDNA sequences and 12 same gene sequences derived from six known Plasmodium parasites and three Babesia parasites. Primary structure of conservative and variable regions of variant sequences was determined also by comparing them with those of six known Plasmodium parasites. To confirm their existence in genome, they were redetected with primers matching their variable regions. PCR systems aimed to roughly detect any eukaryotes and prokaryotes respectively were also applied to search for other pathogens in one of four patients. Results Totally, 19 partial and 23 complete SSU rDNA sequences obtained from four samples. Except eight variant sequences, similarities among sequences from same DNA sample were in general high (more than 98%). The phylogenetic analysis revealed that three cases were infected by P. ovale wallikeri and one by P. ovale curtisi. Four of the variant sequences which obtained from four samples relatively showed high similarities with each other (98.5%-100%). Identical variant sequences actually could be re-obtained from each DNA sample. Their primary structure of conservative and variable regions showed quite fit with that of six known Plasmodium parasites. The test for prokaryote pathogens showed negative and the tests for eukaryotes only found DNA sequences of Human and P. ovale parasites. Conclusion Both P. ovale wallikeri and P. ovale curtisi infections are present in imported malaria cases of China. New type of partial SSU rDNA sequence which assumed to express in a certain life stage of P. ovale was obtained from both P. ovale wallikeri and P. ovale curtisi samples. This discovery would supply information and clues to identify and understand P. ovale parasites more accurately. PMID:24893846

2014-01-01

259

SNPs and MALDI-TOF MS: Tools for DNA Typing in Forensic Paternity Testing and Anthropology  

Microsoft Academic Search

DNA markers used for individual identification in forensic sciences are based on repeat sequences in nuclear DNA and the mitochondrial DNA hypervariable regions 1 and 2. An alternative to these markers is the use of single nucleotide polymorphisms (SNPs). These have a particular advantage in the analysis of degraded or poor samples, which are often all that is available in

Elizabet Petkovski; Christine Keyser-Tracqui; Rémi Hienne; Bertrand Ludes

2005-01-01

260

Evaluation of DNA extraction techniques for detecting Mycobacterium tuberculosis complex organisms in Asian elephant trunk wash samples.  

PubMed

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. PMID:21159933

Kay, Meagan K; Linke, Lyndsey; Triantis, Joni; Salman, M D; Larsen, R Scott

2011-02-01

261

A Mouse Model Uncovers LKB1 as an UVB-Induced DNA Damage Sensor Mediating CDKN1A (p21WAF1/CIP1) Degradation  

PubMed Central

Exposure to ultraviolet (UV) radiation from sunlight accounts for 90% of the symptoms of premature skin aging and skin cancer. The tumor suppressor serine-threonine kinase LKB1 is mutated in Peutz-Jeghers syndrome and in a spectrum of epithelial cancers whose etiology suggests a cooperation with environmental insults. Here we analyzed the role of LKB1 in a UV-dependent mouse skin cancer model and show that LKB1 haploinsufficiency is enough to impede UVB-induced DNA damage repair, contributing to tumor development driven by aberrant growth factor signaling. We demonstrate that LKB1 and its downstream kinase NUAK1 bind to CDKN1A. In response to UVB irradiation, LKB1 together with NUAK1 phosphorylates CDKN1A regulating the DNA damage response. Upon UVB treatment, LKB1 or NUAK1 deficiency results in CDKN1A accumulation, impaired DNA repair and resistance to apoptosis. Importantly, analysis of human tumor samples suggests that LKB1 mutational status could be a prognostic risk factor for UV-induced skin cancer. Altogether, our results identify LKB1 as a DNA damage sensor protein regulating skin UV-induced DNA damage response. PMID:25329316

Esteve-Puig, Rosaura; Gil, Rosa; González-Sánchez, Elena; Bech-Serra, Joan Josep; Grueso, Judit; Hernández-Losa, Javier; Moliné, Teresa; Canals, Francesc; Ferrer, Berta; Cortés, Javier; Bastian, Boris; Ramón y Cajal, Santiago; Martín-Caballero, Juan; Flores, Juana Maria; Vivancos, Ana; García-Patos, Vicenç; Recio, Juan Ángel

2014-01-01

262

Extensive protein and DNA backbone sampling improves structure-based specificity prediction for C2H2 zinc fingers  

PubMed Central

Sequence-specific DNA recognition by gene regulatory proteins is critical for proper cellular functioning. The ability to predict the DNA binding preferences of these regulatory proteins from their amino acid sequence would greatly aid in reconstruction of their regulatory interactions. Structural modeling provides one route to such predictions: by building accurate molecular models of regulatory proteins in complex with candidate binding sites, and estimating their relative binding affinities for these sites using a suitable potential function, it should be possible to construct DNA binding profiles. Here, we present a novel molecular modeling protocol for protein-DNA interfaces that borrows conformational sampling techniques from de novo protein structure prediction to generate a diverse ensemble of structural models from small fragments of related and unrelated protein-DNA complexes. The extensive conformational sampling is coupled with sequence space exploration so that binding preferences for the target protein can be inferred from the resulting optimized DNA sequences. We apply the algorithm to predict binding profiles for a benchmark set of eleven C2H2 zinc finger transcription factors, five of known and six of unknown structure. The predicted profiles are in good agreement with experimental binding data; furthermore, examination of the modeled structures gives insight into observed binding preferences. PMID:21343182

Yanover, Chen; Bradley, Philip

2011-01-01

263

Optimization of minuscule samples for use with cDNA microarrays.  

PubMed

The recent advent of microarray technology and RNA amplification allows us to compare the expression profiles of thousands of genes from small amounts of tissue or cells. We have compared and contrasted various methods of RNA preparation, RNA amplification, target labelling and array analysis in order to achieve a streamlined protocol for microarraying small samples. We have concluded that usage of the NIA 15K cDNA array set, in combination with RNA extraction using the Mini RNA Isolation kit (Zymo), amplification with the RiboAmp kit (Arcturus), followed by indirect labelling via the Atlas PowerScript Fluorescent Labelling kit (using a modified protocol), is optimal with a material derived from either very early stage mouse embryos or individually picked embryonic stem cell colonies. Normalisation using the analysis package Limma (Bioconductor) with data normalisation by print tip Loess, using the "normexp" function with an offset of 50 for background adjustment, and incorporating A-quantile between array normalisation was best with our results. Furthermore, RT-PCR confirmation of array results is achievable without amplification, thereby controlling for amplification bias. These methods will be of great utility in mapping the transcriptome of embryonic and other small samples. PMID:18261801

Hunter, Susan McLean; Mansergh, Fiona C; Evans, Martin J

2008-04-24

264

DNA typing in single cell analysis: Single sperm cells outperform whole genome pre-amplified samples  

Microsoft Academic Search

We recently developed a method in single cell analysis allowing for multiple molecular genetic and cytogenetic analyses of one and the same cell. In the actual study we compared direct DNA typing to DNA typing of whole genome amplified single sperm cells. Showing a PCR efficiency of 62.8%, DNA typing of non-preamplified single sperm cells turned out to be superior

T. Kroneis; I. E. Pickrahn; A. El-Heliebi; G. Schmidt-Gann

265

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

Microsoft Academic Search

Summary Reports indicate an increase in the incidence of DNA fragmentation in male factor infertility and its role in the outcome of assisted reproductive techniques (ART). However, reports are conflicting between the relationships of sperm DNA integrity with conventional semen parameters. We examined the relation- ship between conventional sperm parameters and DNA integrity using acridine orange (AO) test. The study

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

2009-01-01

266

DNA degradation in genetically modified rice with Cry1Ab by food processing methods: Implications for the quantification of genetically modified organisms.  

PubMed

Food processing methods contribute to DNA degradation, thereby affecting genetically modified organism detection and quantification. This study evaluated the effect of food processing methods on the relative transgenic content of genetically modified rice with Cry1Ab. In steamed rice and rice noodles, the levels of Cry1Ab were ?100% and <83%, respectively. Frying and baking in rice crackers contributed to a reduction in Pubi and Cry1Ab, while microwaving caused a decrease in Pubi and an increase in Cry1Ab. The processing methods of sweet rice wine had the most severe degradation effects on Pubi and Cry1Ab. In steamed rice and rice noodles, Cry1Ab was the most stable, followed by SPS and Pubi. However, in rice crackers and sweet rice wine, SPS was the most stable, followed by Cry1Ab and Pubi. Therefore, Cry1Ab is a better representative of transgenic components than is Pubi because the levels of Cry1Ab were less affected compared to Pubi. PMID:25529662

Xing, Fuguo; Zhang, Wei; Selvaraj, Jonathan Nimal; Liu, Yang

2015-05-01

267

Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array–CGH  

PubMed Central

Structural genetic alterations in cancer often involve gene loss or gene amplification. With the advent of microarray approaches for the analysis of the genome, as exemplified by array–CGH (Comparative Genomic Hybridization), scanning for gene-dosage alterations is limited only by issues of DNA microarray density. However, samples of interest to the pathologist often comprise small clusters of just a few hundred cells, which do not provide sufficient DNA for array–CGH analysis. We sought to develop a simple method that would permit amplification of the whole genome without the use of thermocycling or ligation of DNA adaptors, because such a method would lend itself to the automated processing of a large number of tissue samples. We describe a method that permits the isothermal amplification of genomic DNA with high fidelity and limited sequence representation bias. The method is based on strand displacement reactions that propagate by a hyperbranching mechanism, and generate hundreds, or even thousands, of copies of the genome in a few hours. Using whole genome isothermal amplification, in combination with comparative genomic hybridization on cDNA microarrays, we demonstrate the ability to detect gene losses in yeast and gene dosage imbalances in human breast tumor cell lines. Although sequence representation bias in the amplified DNA presents potential problems for CGH analysis, these problems have been overcome by using amplified DNA in both control and tester samples. Gene-dosage alterations of threefold or more can be observed with high reproducibility with as few as 1000 cells of starting material. PMID:12566408

Lage, José M.; Leamon, John H.; Pejovic, Tanja; Hamann, Stefan; Lacey, Michelle; Dillon, Deborah; Segraves, Richard; Vossbrinck, Bettina; González, Antonio; Pinkel, Daniel; Albertson, Donna G.; Costa, Jose; Lizardi, Paul M.

2003-01-01

268

New procedure for recovering extra- and intracellular DNA from marine sediment samples  

NASA Astrophysics Data System (ADS)

Extracellular DNA (eDNA) is a ubiquitous biological compound in aquatic sediment and soil. Despite major methodological advances, analysis of DNA from sediment is still technically challenging, not just because of the co-elution of inhibitory substances, but also due to co-elution of extracellular DNA, which potentially leads to an overestimate of the actual diversity. Previous studies suggested that eDNA might play an important role in biogeochemical element cycling, horizontal gene transfer and stabilization of biofilm structures. Several protocols based on the precipitation of eDNA e.g. with CTAB and ethanol have already been published. However, using these methods we did not succeed in quantifying very low amounts of eDNA (e.g. <1?g eDNA/g dry wt) in marine sediment even when using DNA carriers like glycogen. Since the recovery of eDNA by precipitation strongly depends on its concentration, these previously published procedures are not adequate for deep biosphere sediment due to the low eDNA content. We have focused on the question whether eDNA could be a source of nitrogen and phosphorus for microbes in the subseafloor biosphere. Therefore we developed a new method for the (semi)-quantitative extraction of eDNA from sediment. The new extraction procedure is based on sequential washing of the sediment to remove simultaneously eDNA and microbial cells without lysing them. After separation of the cells by centrifugation, the eDNA was extracted from the supernatant and purified by adsorption onto a solid phase, followed by removal of the solids and subsequent elution of the pure eDNA. Intracellular DNA (iDNA) was extracted and purified from the cell pellet using a commercial DNA extraction kit. Additional to a very low detection limit and reproducible quantification, this new method allows separation and purification of both extracellular and intracellular DNA to an extent that inhibitors are removed and downstream applications like PCR can be performed. To evaluate the new extraction method two sediments with rather opposing composition were analyzed. Sediment from the South Pacific Gyre, the most oligotrophic oceanic region on earth and organic-rich Baltic Sea sediment (Northern Germany) were processed. Using this new procedure high purity genomic iDNA and eDNA with a molecular size range between 20 bp and 50k bp can be simultaneously recovered even from very oligotrophic sediment with very low cell abundances. The main fraction of recovered eDNA was suitable for downstream applications like PCR and had a molecular size that indicates minimal shearing. Despite about two decades of research many questions about deep subsurface life remain unanswered. The fact that microbes can be found even in deep oligotrophic marine sediment raises the fundamental questions of the types and availability of substrates and their biogeochemical cycling. This is the first study that provides evidence that eDNA is an important potential substrate for microorganisms in the deep biosphere. Also, our results show a link between cell counts and eDNA content, indicating that the eDNA pool in the investigated sediment consist mainly of microbial DNA. Comparative sequence analysis of extracted iDNA and eDNA will provide deeper insights into the origin and turnover of eDNA and the apparent microbial community composition in the deep biosphere.

Alawi, M.; Kallmeyer, J.

2012-12-01

269

Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing  

PubMed Central

Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy. PMID:24039948

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

270

Selection and Application of ssDNA Aptamers to Detect Active TB from Sputum Samples  

PubMed Central

Background Despite the enormous global burden of tuberculosis (TB), conventional approaches to diagnosis continue to rely on tests that have major drawbacks. The improvement of TB diagnostics relies, not only on good biomarkers, but also upon accurate detection methodologies. The 10-kDa culture filtrate protein (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) are potent T-cell antigens that are recognised by over 70% of TB patients. Aptamers, a novel sensitive and specific class of detection molecules, has hitherto, not been raised to these relatively TB-specific antigens. Methods DNA aptamers that bind to the CFP-10.ESAT-6 heterodimer were isolated. To assess their affinity and specificity to the heterodimer, aptamers were screened using an enzyme-linked oligonucleotide assay (ELONA). One suitable aptamer was evaluated by ELONA using sputum samples obtained from 20 TB patients and 48 control patients (those with latent TB infection, symptomatic non TB patients, and healthy laboratory volunteers). Culture positivity for Mycobacterium tuberculosis (Mtb) served as the reference standard. Accuracy and cut-points were evaluated using ROC curve analysis. Results Twenty-four out of the 66 aptamers that were isolated bound significantly (p<0.05) to the CFP-10.ESAT-6 heterodimer and six were further evaluated. Their dissociation constant (KD) values were in the nanomolar range. One aptamer, designated CSIR 2.11, was evaluated using sputum samples. CSIR 2.11 had sensitivity and specificity of 100% and 68.75% using Youden’s index and 35% and 95%, respectively, using a rule-in cut-point. Conclusion This preliminary proof-of-concept study suggests that a diagnosis of active TB using anti-CFP-10.ESAT-6 aptamers applied to human sputum samples is feasible. PMID:23056492

Rotherham, Lia S.; Maserumule, Charlotte; Dheda, Keertan; Theron, Jacques; Khati, Makobetsa

2012-01-01

271

CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3  

Microsoft Academic Search

The prokaryotic CRISPR\\/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease\\/helicase are required for CRISPR interference in Escherichia coli, but it is unknown how natural target DNA molecules

E. R. Westra; P. B. G. Erp; S. P. Wong; T. Künne; C. L. C. Seegers; S. Bollen; M. M. Jore; Vos de W. M; R. T. Dame; Vries de R; S. J. J. Brouns; Oost van der J

2012-01-01

272

Cellulose degradation by micromonosporas recovered from freshwater lakes and classification of these actinomycetes by DNA gyrase B gene sequencing.  

PubMed

A number of Micromonospora strains isolated from the water column, sediment, and cellulose baits placed in freshwater lakes were shown to be able to degrade cellulose in lake water without any addition of nutrients. A selective isolation method was also developed to demonstrate that CFU arose from both spores and hyphae that inhabit the lake environment. Gyrase B gene sequencing performed on the isolates identified a number of new centers of variation within Micromonospora, but the most actively cellulolytic strains were recovered in a single cluster that equated with the type species of the genus, M. chalcea. PMID:18820070

de Menezes, Alexandre B; Lockhart, Robert J; Cox, Michael J; Allison, Heather E; McCarthy, Alan J

2008-11-01

273

Cellulose Degradation by Micromonosporas Recovered from Freshwater Lakes and Classification of These Actinomycetes by DNA Gyrase B Gene Sequencing?  

PubMed Central

A number of Micromonospora strains isolated from the water column, sediment, and cellulose baits placed in freshwater lakes were shown to be able to degrade cellulose in lake water without any addition of nutrients. A selective isolation method was also developed to demonstrate that CFU arose from both spores and hyphae that inhabit the lake environment. Gyrase B gene sequencing performed on the isolates identified a number of new centers of variation within Micromonospora, but the most actively cellulolytic strains were recovered in a single cluster that equated with the type species of the genus, M. chalcea. PMID:18820070

de Menezes, Alexandre B.; Lockhart, Robert J.; Cox, Michael J.; Allison, Heather E.; McCarthy, Alan J.

2008-01-01

274

A quantitative evaluation of two methods for preserving hair samples  

USGS Publications Warehouse

Hair samples are an increasingly important DNA source for wildlife studies, yet optimal storage methods and DNA degradation rates have not been rigorously evaluated. We tested amplification success rates over a one-year storage period for DNA extracted from brown bear (Ursus arctos) hair samples preserved using silica desiccation and -20 ??C freezing. For three nuclear DNA microsatellites, success rates decreased significantly after a six-month time point, regardless of storage method. For a 1000 bp mitochondrial fragment, a similar decrease occurred after a two-week time point. Minimizing delays between collection and DNA extraction will maximize success rates for hair-based noninvasive genetic sampling projects.

Roon, D.A.; Waits, L.P.; Kendall, K.C.

2003-01-01

275

Simultaneous assessment of the macrobiome and microbiome in a bulk sample of tropical arthropods through DNA metasystematics  

PubMed Central

Conventional assessments of ecosystem sample composition are based on morphology-based or DNA barcode identification of individuals. Both approaches are costly and time-consuming, especially when applied to the large number of specimens and taxa commonly included in ecological investigations. Next-generation sequencing approaches can overcome the bottleneck of individual specimen isolation and identification by simultaneously sequencing specimens of all taxa in a bulk mixture. Here we apply multiple parallel amplification primers, multiple DNA barcode markers, 454-pyrosequencing, and Illumina MiSeq sequencing to the same sample to maximize recovery of the arthropod macrobiome and the bacterial and other microbial microbiome of a bulk arthropod sample. We validate this method with a complex sample containing 1,066 morphologically distinguishable arthropods from a tropical terrestrial ecosystem with high taxonomic diversity. Multiamplicon next-generation DNA barcoding was able to recover sequences corresponding to 91% of the distinguishable individuals in a bulk environmental sample, as well as many species present as undistinguishable tissue. 454-pyrosequencing was able to recover 10 more families of arthropods and 30 more species than did conventional Sanger sequencing of each individual specimen. The use of other loci (16S and 18S ribosomal DNA gene regions) also added the detection of species of microbes associated with these terrestrial arthropods. This method greatly decreases the time and money necessary to perform DNA-based comparisons of biodiversity among ecosystem samples. This methodology opens the door to much cheaper and increased capacity for ecological and evolutionary studies applicable to a wide range of socio-economic issues, as well as a basic understanding of how the world works. PMID:24808136

Gibson, Joel; Shokralla, Shadi; Porter, Teresita M.; King, Ian; van Konynenburg, Steven; Janzen, Daniel H.; Hallwachs, Winnie; Hajibabaei, Mehrdad

2014-01-01

276

Fungal DNA Detected in Blood Samples of Patients Who Received Contaminated Methylprednisolone Injections Reveals Increased Complexity of Causative Agents  

PubMed Central

Using Exserohilum rostratum-specific and panfungal real-time PCR, we studied 24 blood samples and 2 synovial fluid specimens from 20 patients with persistent or worsening pain following injections of contaminated methylprednisolone. Seven blood specimens from 6 patients were significantly positive for fungal DNA by panfungal PCR, with multiple fungal species identified. PMID:24719442

Zhao, Yanan; Armeanu, Emilian; DiVerniero, Richard; Lewis, Terri A.; Dobson, Richard C.; Kontoyiannis, Dimitrios P.; Roilides, Emmanuel; Walsh, Thomas J.

2014-01-01

277

Putative odorant-degrading esterase cDNA from the moth Mamestra brassicae: cloning and expression patterns in male and female antennae.  

PubMed

An esterase cDNA was isolated from the cabbage armyworm Mamestra brassicae antennae by PCR strategy. The full-length cDNA, designated as Mbra-EST, contains a 1638 bp open reading frame encoding a predicted protein of 546 amino acids. This predicted protein presents the structural characteristics of known insect carboxyl-esterases, in particular the Ser-His-Glu catalytic triad. The expression pattern of the gene was studied by RT-PCR, Northern-blot and in situ hybridization. The ribosomal protein rpL8 gene from M. brassicae was also cloned to obtain a normalized tool for the comparative gene expression studies. Mbra-EST transcripts are specifically expressed in the antennae of males and females and in the proboscis of males. In antennae of both sexes, expression is restricted to the olfactory sensilla trichodea, suggesting a role in degradation of odorant acetate compounds, such as pheromones as well as plant volatile acetate components. PMID:15201205

Maïbèche-Coisne, Martine; Merlin, Christine; François, Marie-Christine; Queguiner, Isabelle; Porcheron, Patrick; Jacquin-Joly, Emmanuelle

2004-06-01

278

Effects of ascorbic acid on sperm motility, viability, acrosome reaction and DNA integrity in teratozoospermic samples  

PubMed Central

Background: Oxidative stress in teratozoospermic semen samples caused poor assisted reproductive techniques (ART) outcomes. Among antioxidants, ascorbic acid is a naturally occurring free radical scavenger and as such its presence assists various other mechanisms in decreasing numerous disruptive free radical processes. Objective: The main goal of this study was to evaluate potential protective effects of ascorbic acid supplementation during in vitro culture of teratozoospermic specimens. Materials and Methods: Teratozoospermic semen samples that collected from 15 volunteers were processed, centrifuged and incubated at 37oC until sperm swimmed-up. Supernatant was divided into four groups and incubated at 37oC for one hour under different experimental conditions: Control, 10 µm A23187, 600µm ascorbic acid and 10 µm A23187+600 µm ascorbic acid. After incubation sperm motility, viability, acrosome reaction, DNA damage and malondialdehyde levels were evaluated. Results: Our results indicated that after one hour incubation, ascorbic acid significantly reduced malondialdehyde level in ascorbic acid group (1.4±0.11 nmol/ml) compared to control group (1.58±0.13 nmol/ml) (p<0.001). At the end of incubation, progressive motility and viability in ascorbic acid group (64.5±8.8% and 80.3±6.4%, respectively) were significantly (p<0.05 and p<0.001, respectively) higher than the control group (54.5±6.8% and 70.9±7.3%, respectively). A23187 significantly (p<0.0001) increased acrosome reaction in A23187 group (37.3±5.6%) compared to control group (8.5±3.2%) and this effect of A23187 attenuated by ascorbic acid in ascorbic acid+A23187 group (17.2±4.4%). DNA fragmentation in ascorbic acid group (20±4.1%) was significantly (p<0.001) lower than controls (28.9±4.6%). Conclusion: In vitro ascorbic acid supplementation during teratozoospermic semen processing for ART could protect teratozoospermic specimens against oxidative stress, and it could improve ART outcome. PMID:24799867

Fanaei, Hamed; Khayat, Samira; Halvaei, Iman; Ramezani, Vahid; Azizi, Yaser; Kasaeian, Amir; Mardaneh, Jalal; Parvizi, Mohammad Reza; Akrami, Maryam

2014-01-01

279

DNA sequences identical to Pneumocystis carinii f. sp. carinii and Pneumocystis carinii f. sp. hominis in samples of air spora.  

PubMed Central

Samples of ambient air collected with three different types of spore traps in a rural location were examined for the presence of Pneumocystis carinii by screening for P. carinii-specific DNA sequences by DNA amplification. Eleven spore trap samples were analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial large subunit rRNA of P. carinii f. sp. carinii and P. carinii f. sp. hominis. The samples were collected over a 3-year period during the months of May to September, with a range of sampling times from 9 to 240 h. One air sample from an animal facility housing P. carinii-infected rats was also examined. P. carinii-specific amplification products were obtained from samples from each of the spore traps. The amplification products from eight air samples were cloned and sequenced. The majority of the recombinants from each of these samples had sequences identical to those of P. carinii f. sp. carinii and P. carinii f. sp. hominis, and a number of clones had single-base differences. These data suggest that sequences identical to those of P. carinii f. sp. carinii and P. carinii f. sp. hominis can be detected in samples of air collected in a rural location and that P. carinii may be a component of the air spora of rural Oxfordshire. PMID:8784583

Wakefield, A E

1996-01-01

280

DNA  

ERIC Educational Resources Information Center

This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

Stent, Gunther S.

1970-01-01

281

An improved protocol for DNA extraction from alkaline soil and sediment samples for constructing metagenomic libraries.  

PubMed

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. PMID:21519906

Verma, Digvijay; Satyanarayana, T

2011-09-01

282

Commercial DNA extraction kits impact observed microbial community composition in permafrost samples.  

PubMed

The total community genomic DNA (gDNA) from permafrost was extracted using four commercial DNA extraction kits. The gDNAs were compared using quantitative real-time PCR (qPCR) targeting 16S rRNA genes and bacterial diversity analyses obtained via 454 pyrosequencing of the 16S rRNA (V3 region) amplified in single or nested PCR. The FastDNA(®) SPIN (FDS) Kit provided the highest gDNA yields and 16S rRNA gene concentrations, followed by MoBio PowerSoil(®) (PS) and MoBio PowerLyzer™ (PL) kits. The lowest gDNA yields and 16S rRNA gene concentrations were from the Meta-G-Nome™ (MGN) DNA Isolation Kit. Bacterial phyla identified in all DNA extracts were similar to that found in other soils and were dominated by Actinobacteria, Firmicutes, Gemmatimonadetes, Proteobacteria, and Acidobacteria. Weighted UniFrac and statistical analyses indicated that bacterial community compositions derived from FDS, PS, and PL extracts were similar to each other. However, the bacterial community structure from the MGN extracts differed from other kits exhibiting higher proportions of easily lysed ?- and ?-Proteobacteria and lower proportions of Actinobacteria and Methylocystaceae important in carbon cycling. These results indicate that gDNA yields differ between the extraction kits, but reproducible bacterial community structure analysis may be accomplished using gDNAs from the three bead-beating lysis extraction kits. PMID:24102625

Vishnivetskaya, Tatiana A; Layton, Alice C; Lau, Maggie C Y; Chauhan, Archana; Cheng, Karen R; Meyers, Arthur J; Murphy, Jasity R; Rogers, Alexandra W; Saarunya, Geetha S; Williams, Daniel E; Pfiffner, Susan M; Biggerstaff, John P; Stackhouse, Brandon T; Phelps, Tommy J; Whyte, Lyle; Sayler, Gary S; Onstott, Tullis C

2014-01-01

283

The effect of dilution and the use of a post-extraction nucleic acid purification column on the accuracy, precision, and inhibition of environmental DNA samples  

USGS Publications Warehouse

Isolation of environmental DNA (eDNA) is an increasingly common method for detecting presence and assessing relative abundance of rare or elusive species in aquatic systems via the isolation of DNA from environmental samples and the amplification of species-specific sequences using quantitative PCR (qPCR). Co-extracted substances that inhibit qPCR can lead to inaccurate results and subsequent misinterpretation about a species’ status in the tested system. We tested three treatments (5-fold and 10-fold dilutions, and spin-column purification) for reducing qPCR inhibition from 21 partially and fully inhibited eDNA samples collected from coastal plain wetlands and mountain headwater streams in the southeastern USA. All treatments reduced the concentration of DNA in the samples. However, column purified samples retained the greatest sensitivity. For stream samples, all three treatments effectively reduced qPCR inhibition. However, for wetland samples, the 5-fold dilution was less effective than other treatments. Quantitative PCR results for column purified samples were more precise than the 5-fold and 10-fold dilutions by 2.2× and 3.7×, respectively. Column purified samples consistently underestimated qPCR-based DNA concentrations by approximately 25%, whereas the directional bias in qPCR-based DNA concentration estimates differed between stream and wetland samples for both dilution treatments. While the directional bias of qPCR-based DNA concentration estimates differed among treatments and locations, the magnitude of inaccuracy did not. Our results suggest that 10-fold dilution and column purification effectively reduce qPCR inhibition in mountain headwater stream and coastal plain wetland eDNA samples, and if applied to all samples in a study, column purification may provide the most accurate relative qPCR-based DNA concentrations estimates while retaining the greatest assay sensitivity.

Mckee, Anna M.; Spear, Stephen F.; Pierson, Todd W.

2014-01-01

284

A filter paper-based microdevice for low-cost, rapid, and automated DNA extraction and amplification from diverse sample types.  

PubMed

A plastic microfluidic device that integrates a filter disc as a DNA capture phase was successfully developed for low-cost, rapid and automated DNA extraction and PCR amplification from various raw samples. The microdevice was constructed by sandwiching a piece of Fusion 5 filter, as well as a PDMS (polydimethylsiloxane) membrane, between two PMMA (poly(methyl methacrylate)) layers. An automated DNA extraction from 1 ?L of human whole blood can be finished on the chip in 7 minutes by sequentially aspirating NaOH, HCl, and water through the filter. The filter disc containing extracted DNA was then taken out directly for PCR. On-chip DNA purification from 0.25-1 ?L of human whole blood yielded 8.1-21.8 ng of DNA, higher than those obtained using QIAamp® DNA Micro kits. To realize DNA extraction from raw samples, an additional sample loading chamber containing a filter net with an 80 ?m mesh size was designed in front of the extraction chamber to accommodate sample materials. Real-world samples, including whole blood, dried blood stains on Whatman® 903 paper, dried blood stains on FTA™ cards, buccal swabs, saliva, and cigarette butts, can all be processed in the system in 8 minutes. In addition, multiplex amplification of 15 STR (short tandem repeat) loci and Sanger-based DNA sequencing of the 520 bp GJB2 gene were accomplished from the filters that contained extracted DNA from blood. To further prove the feasibility of integrating this extraction method with downstream analyses, "in situ" PCR amplifications were successfully performed in the DNA extraction chamber following DNA purification from blood and blood stains without DNA elution. Using a modified protocol to bond the PDMS and PMMA, our plastic PDMS devices withstood the PCR process without any leakage. This study represents a significant step towards the practical application of on-chip DNA extraction methods, as well as the development of fully integrated genetic analytical systems. PMID:25070548

Gan, Wupeng; Zhuang, Bin; Zhang, Pengfei; Han, Junping; Li, Cai-Xia; Liu, Peng

2014-10-01

285

From sample to PCR product in under 45 minutes: a polymeric integrated microdevice for clinical and forensic DNA analysis.  

PubMed

The extraction and amplification of DNA from biological samples is laborious and time-consuming, requiring numerous instruments and sample handling steps. An integrated, single-use, poly(methyl methacrylate) (PMMA) microdevice for DNA extraction and amplification would benefit clinical and forensic communities, providing a completely closed system with rapid sample-in-PCR-product-out capability. Here, we show the design and simple flow control required for enzyme-based DNA preparation and PCR from buccal swabs or liquid whole blood samples with an ~5-fold reduction in time. A swab containing cells or DNA could be loaded into a novel receptacle together with the DNA liberation reagents, heated using an infrared heating system, mixed with PCR reagents for one of three different target sets under syringe-driven flow, and thermally-cycled in less than 45 min, an ~6-fold reduction in analysis time as compared to conventional methods. The 4 : 1 PCR reagents : DNA ratio required to provide the correct final concentration of all PCR components for effective amplification was verified using image analysis of colored dyes in the PCR chamber. Novel single-actuation, 'normally-open' adhesive valves were shown to effectively seal the PCR chamber during thermal cycling, preventing air bubble expansion. The effectiveness of the device was demonstrated using three target sets: the sex-typing gene Amelogenin, co-amplification of the ?-globin and gelsolin genes, and the amplification of 15 short tandem repeat (STR) loci plus Amelogenin. The use of the integrated microdevice was expanded to the analysis of liquid blood samples which, when incubated with the DNA liberation reagents, form a brown precipitate that inhibits PCR. A simple centrifugation of the integrated microchips (on a custom centrifuge), mobilized the precipitate away from the microchannel entrance, improving amplification of the ?-globin and gelsolin gene fragments by ~6-fold. This plastic integrated microdevice represents a microfluidic platform with potential for evolution into point-of-care prototypes for application to both clinical and forensic analyses, providing a 5-fold reduction from conventional analysis time. PMID:23389252

Lounsbury, Jenny A; Karlsson, Anne; Miranian, Daniel C; Cronk, Stephen M; Nelson, Daniel A; Li, Jingyi; Haverstick, Doris M; Kinnon, Paul; Saul, David J; Landers, James P

2013-04-01

286

Forensic Analysis of Canine DNA Samples in the Undergraduate Biochemistry Laboratory  

ERIC Educational Resources Information Center

Recent advances in canine genomics have allowed the development of highly distinguishing methods of analysis for both nuclear and mitochondrial DNA. We describe a laboratory exercise suitable for an undergraduate biochemistry course in which the polymerase chain reaction is used to amplify hypervariable regions of DNA from dog hair and saliva…

Carson, Tobin M.; Bradley, Sharonda Q.; Fekete, Brenda L.; Millard, Julie T.; LaRiviere, Frederick J.

2009-01-01

287

Mre11-dependent degradation of stalled DNA replication forks is prevented by BRCA2 and PARP1.  

PubMed

PARP inhibitors are currently being used in clinical trials to treat BRCA1- or BRCA2-defective tumors, based on the synthetic lethal interaction between PARP1 and BRCA1/2-mediated homologous recombination (HR). However, the molecular mechanisms that drive this synthetic lethality remain unclear. Here, we show increased levels of Mre11, a key component of MRN (Mre11-Rad50-Nbs1) complex that plays a role in the restart of stalled replication forks and enhanced resection at stalled replication forks in BRCA2-deficient cells. BRCA2-deficient cells also showed hypersensitivity to the Mre11 inhibitor mirin. Interestingly, PARP1 activity was required to protect stalled forks from Mre11-dependent degradation. Resistance to PARP inhibition in BRCA2-mutant cells led to reduced levels of Mre11 foci and also rescued their sensitivity to mirin. Taken together, our findings not only show that Mre11 activity is required for the survival of BRCA2 mutant cells but also elucidate roles for both the BRCA2 and PARP1 proteins in protecting stalled replication forks, which offers insight into the molecular mechanisms of the synthetic lethality between BRCA2 and PARP1. PMID:22447567

Ying, Songmin; Hamdy, Freddie C; Helleday, Thomas

2012-06-01

288

Simultaneous Detection of Multiple DNA Adducts in Human Lung Samples by Isotope-Dilution UPLC-MS/MS.  

PubMed

Recent studies have demonstrated that various DNA adducts can be detected in human tissues and fluids using liquid chromatography connected to tandem mass spectrometry (LC-MS/MS). However, the utility of a single DNA adduct as a biomarker in risk assessment is debatable because humans are exposed to many genotoxicants. We established a method to measure DNA adducts derived from 16 ubiquitous genotoxicants and developed an analytical technique for their simultaneous quantification by ultra performance liquid chromatography (UPLC)-MS/MS. Methods for the enrichment of the analytes from DNA hydrolysates and chromatographic separation preceding mass spectrometric analysis were optimized, and the resultant technique was used for the simultaneous analysis of the 16 DNA adducts in human lung biopsy specimens. Eleven adducts (formed by benzo[a]pyrene, 1-methylpyrene, 4-aminobiphenyl, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 1-methoxy-3-indolylmethylglucosinolate, 5-hydroxymethylfurfural, and malondialdehyde) were not detected in any tissue sample (limits of detection: 0.02-7.1 adducts/10(8) nucleosides). 3,N(4)-etheno-2'-deoxycytidine and 1,N(6)-etheno-2'-deoxyadenosine, formed from 2,3-epoxyaldehydes of endogenous lipid peroxidation products, were present in all subjects (16.9-115.3 and 27.2-179/10(8) nucleosides, respectively). The same was true for N(2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine, the major adduct of methyleugenol (1.7-23.7/10(8) nucleosides). A minor adduct of methyleugenol and two adducts of furfuryl alcohol were detected in several pulmonary specimens. Taken together, we developed a targeted approach for the simultaneous mass spectrometric analyses of 16 DNA adducts, which can be easily extended by adducts formed from other mutagens. The method allowed one to detect adducts of furfuryl alcohol and methyleugenol in samples of human lung. PMID:25423194

Monien, Bernhard H; Schumacher, Fabian; Herrmann, Kristin; Glatt, Hansruedi; Turesky, Robert J; Chesné, Christophe

2014-12-01

289

Monitoring the degradation of a thermally aged EPDM terpolymer by 1H NMR relaxation measurements of solvent swelled samples  

Microsoft Academic Search

1H nuclear magnetic resonance (NMR) relaxation times were investigated as a method for monitoring the degradation of polymeric materials. The properties of an ethylene–propylene–diene (EPDM) terpolymer, oven aged at 140°C, were first characterized by traditional mechanical and solution measurements including ultimate tensile elongation, tensile strength, tensile modulus, gel fraction, solvent uptake and density. The elongation and density results provided a

Roger A. Assink; Kenneth T. Gillen; Briana Sanderson

2002-01-01

290

Increased presence of Epstein-Barr virus DNA in ocular fluid samples from HIV negative immunocompromised patients with uveitis  

PubMed Central

AIMS—To investigate whether routine testing for Epstein-Barr virus (EBV) is necessary in the examination of a patient with uveitis.?METHODS—Intraocular EBV DNA was determined in 183 ocular fluid samples taken from patients with AIDS and uveitis, HIV negative immunocompromised uveitis, acute retinal necrosis, toxoplasma chorioretinitis, intraocular lymphoma, anterior uveitis, and miscellaneous uveitis of unknown cause. In 82 samples from this group of patients paired serum/ocular fluid analysis was performed to detect local antibody production against EBV. Controls (n=46) included ocular fluid samples taken during surgery for diabetic retinopathy, macular pucker, or cataract.?RESULTS—Serum antibody titres to EBV capsid antigen proved to be significantly increased in HIV negative immunocompromised patients with uveitis (p<0.01) compared with controls. Local antibody production revealed only three positive cases out of 82 patients tested, two results were borderline positive and one patient had uveitis caused by VZV. EBV DNA was detected in three out of 46 control ocular fluid samples. In the different uveitis groups EBV DNA was noted, but was not significantly higher than in the controls, except in six out of 11 HIV negative immunocompromised patients (p=0.0008). In four out of these six cases another infectious agent (VZV, HSV, CMV, or Toxoplasma gondii) had previously been identified as the cause of the uveitis.?CONCLUSIONS—When comparing various groups of uveitis patients, EBV DNA was found more often in HIV negative immunocompromised patients with uveitis. Testing for EBV does not have to be included in the routine management of patients with uveitis, since indications for an important role of this virus were not found in the pathogenesis of intraocular inflammation.?? Keywords: Epstein-Barr virus; intraocular fluid; polymerase chain reaction; uveitis PMID:9602620

Ongkosuwito, J.; Van der Lelij, A.; Bruinenberg, M.; Doorn, M. W.; Feron, E.; Hoyng, C.; de Keizer, R. J W; Klok, A.; Kijlstra, A.

1998-01-01

291

Sensitivity testing of trypanosome detection by PCR from whole blood samples using manual and automated DNA extraction methods.  

PubMed

Automated extraction of DNA for testing of laboratory samples is an attractive alternative to labour-intensive manual methods when higher throughput is required. However, it is important to maintain the maximum detection sensitivity possible to reduce the occurrence of type II errors (false negatives; failure to detect the target when it is present), especially in the biomedical field, where PCR is used for diagnosis. We used blood infected with known concentrations of Trypanosoma copemani to test the impact of analysis techniques on trypanosome detection sensitivity by PCR. We compared combinations of a manual and an automated DNA extraction method and two different PCR primer sets to investigate the impact of each on detection levels. Both extraction techniques and specificity of primer sets had a significant impact on detection sensitivity. Samples extracted using the same DNA extraction technique performed substantially differently for each of the separate primer sets. Type I errors (false positives; detection of the target when it is not present), produced by contaminants, were avoided with both extraction methods. This study highlights the importance of testing laboratory techniques with known samples to optimise accuracy of test results. PMID:25124940

Dunlop, J; Thompson, C K; Godfrey, S S; Thompson, R C A

2014-11-01

292

Concentrations of Glyphosate, Its Degradation Product, Aminomethylphosphonic Acid, and Glufosinate in Ground- and Surface-Water, Rainfall, and Soil Samples Collected in the United States, 2001-06  

USGS Publications Warehouse

The U.S. Geological Survey conducted a number of studies from 2001 through 2006 to investigate and document the occurrence, fate, and transport of glyphosate, its degradation product, aminomethylphosphonic acid (AMPA), and glufosinate in 2,135 ground- and surface-water samples, 14 rainfall samples, and 193 soil samples. Analytical methods were developed to detect and measure glyphosate, AMPA, and glufosinate in water, rainfall, and soil. Results show that AMPA was detected more frequently and occurred at similar or higher concentrations than the parent compound, glyphosate, whereas glufosinate was seldom found in the environment. Glyphosate and AMPA were detected more frequently in surface water than in ground water. Trace levels of glyphosate and AMPA may persist in the soil from year to year. The methods and data described in this report are useful to researchers and regulators interested in the occurrence, fate, and transport of glyphosate and AMPA in the environment.

Scribner, Elisabeth A.; Battaglin, William A.; Gilliom, Robert J.; Meyer, Michael T.

2007-01-01

293

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

E-print Network

UV irradiation damages DNA and activates expression of genes encoding proteins helpful for survival under DNA stress. These proteins are often deleterious in the absence of DNA damage. Here, we investigate mechanisms used ...

Pruteanu, Mihaela

294

Glycogen Synthase Kinase (GSK) 3? Phosphorylates and Protects Nuclear Myosin 1c from Proteasome-Mediated Degradation to Activate rDNA Transcription in Early G1 Cells  

PubMed Central

Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3? phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3? selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3??/? mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3? directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3?-mediated phosphorylation of NM1 is required for pol I transcription activation. PMID:24901984

Sarshad, Aishe A.; Corcoran, Martin; Al-Muzzaini, Bader; Borgonovo-Brandter, Laura; Von Euler, Anne; Lamont, Douglas; Visa, Neus; Percipalle, Piergiorgio

2014-01-01

295

Auxin-induced Rapid Degradation of Inhibitor of Caspase-activated DNase (ICAD) Induces Apoptotic DNA Fragmentation, Caspase Activation, and Cell Death  

PubMed Central

Caspase-activated DNase (CAD) is a major apoptotic nuclease, responsible for DNA fragmentation and chromatin condensation during apoptosis. CAD is normally activated in apoptosis as a result of caspase cleavage of its inhibitory chaperone ICAD. Other aspects of CAD regulation are poorly understood. In particular, it has been unclear whether direct CAD activation in non-apoptotic living cells can trigger cell death. Taking advantage of the auxin-inducible degron (AID) system, we have developed a suicide system with which ICAD is rapidly degraded in living cells in response to the plant hormone auxin. Our studies demonstrate that rapid ICAD depletion is sufficient to activate CAD and induce cell death in DT40 and yeast cells. In the vertebrate cells, ectopic CAD activation triggered caspase activation and subsequent hallmarks of caspase-dependent apoptotic changes, including phosphatidylserine exposure and nuclear fragmentation. These observations not only suggest that CAD activation drives apoptosis through a positive feedback loop, but also identify a unique suicide system that can be used for controlling gene-modified organisms. PMID:25248749

Samejima, Kumiko; Ogawa, Hiromi; Ageichik, Alexander V.; Peterson, Kevin L.; Kaufmann, Scott H.; Kanemaki, Masato T.; Earnshaw, William C.

2014-01-01

296

A novel immunity system for bacterial nucleic acid degrading toxins and its recruitment in various eukaryotic and DNA viral systems  

PubMed Central

The use of nucleases as toxins for defense, offense or addiction of selfish elements is widely encountered across all life forms. Using sensitive sequence profile analysis methods, we characterize a novel superfamily (the SUKH superfamily) that unites a diverse group of proteins including Smi1/Knr4, PGs2, FBXO3, SKIP16, Syd, herpesviral US22, IRS1 and TRS1, and their bacterial homologs. Using contextual analysis we present evidence that the bacterial members of this superfamily are potential immunity proteins for a variety of toxin systems that also include the recently characterized contact-dependent inhibition (CDI) systems of proteobacteria. By analyzing the toxin proteins encoded in the neighborhood of the SUKH superfamily we predict that they possess domains belonging to diverse nuclease and nucleic acid deaminase families. These include at least eight distinct types of DNases belonging to HNH/EndoVII- and restriction endonuclease-fold, and RNases of the EndoU-like and colicin E3-like cytotoxic RNases-folds. The N-terminal domains of these toxins indicate that they are extruded by several distinct secretory mechanisms such as the two-partner system (shared with the CDI systems) in proteobacteria, ESAT-6/WXG-like ATP-dependent secretory systems in Gram-positive bacteria and the conventional Sec-dependent system in several bacterial lineages. The hedgehog-intein domain might also release a subset of toxic nuclease domains through auto-proteolytic action. Unlike classical colicin-like nuclease toxins, the overwhelming majority of toxin systems with the SUKH superfamily is chromosomally encoded and appears to have diversified through a recombination process combining different C-terminal nuclease domains to N-terminal secretion-related domains. Across the bacterial superkingdom these systems might participate in discriminating `self’ or kin from `non-self’ or non-kin strains. Using structural analysis we demonstrate that the SUKH domain possesses a versatile scaffold that can be used to bind a wide range of protein partners. In eukaryotes it appears to have been recruited as an adaptor to regulate modification of proteins by ubiquitination or polyglutamylation. Similarly, another widespread immunity protein from these toxin systems, namely the suppressor of fused (SuFu) superfamily has been recruited for comparable roles in eukaryotes. In animal DNA viruses, such as herpesviruses, poxviruses, iridoviruses and adenoviruses, the ability of the SUKH domain to bind diverse targets has been deployed to counter diverse anti-viral responses by interacting with specific host proteins. PMID:21306995

Zhang, Dapeng; Iyer, Lakshminarayan M.; Aravind, L.

2011-01-01

297

Identification of sample-specific sequences in mammalian cDNA and genomic DNA by the novel ligation-mediated subtraction (Limes)  

PubMed Central

The representational difference analysis (RDA) and other subtraction techniques are used to enrich sample-specific sequences by elimination of ubiquitous sequences existing in both the sample of interest (tester) and the subtraction partner (driver). While applying the RDA to genomic DNA of cutaneous lymphoma cells in order to identify tumor relevant alterations, we predominantly isolated repetitive sequences and artificial repeat-mediated fusion products of otherwise independent PCR fragments (PCR hybrids). Since these products severely interfered with the isolation of tester-specific fragments, we developed a considerably more robust and efficient approach, termed ligation-mediated subtraction (Limes). In first applications of Limes, genomic sequences and/or transcripts of genes involved in the regulation of transcription, such as transforming growth factor ? stimulated clone 22 related gene (TSC-22R), cell death and cytokine production (caspase-1) or antigen presentation (HLA class II sequences), were found to be completely absent in a cutaneous lymphoma line. On the assumption that mutations in tumor-relevant genes can affect their transcription pattern, a protocol was developed and successfully applied that allows the identification of such sequences. Due to these results, Limes may substitute/supplement other subtraction/comparison techniques such as RDA or DNA microarray techniques in a variety of different research fields. PMID:11160940

Hansen-Hagge, Thomas E.; Trefzer, Uwe; zu Reventlow, Anna Sophie; Kaltoft, Keld; Sterry, Wolfram

2001-01-01

298

The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From these results, we conclude that the cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting the ubiquitin-proteasome dependent degradation of XRCC1 in an Epac-dependent pathway in lung cancer cells.

Cho, Eun-Ah [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)] [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)] [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

2012-06-01

299

Detection of environmental DNA of Bigheaded Carps in samples collected from selected locations in the St. Croix River and in the Mississippi River  

USGS Publications Warehouse

The use of molecular methods, such as the detection of environmental deoxyribonucleic acid (eDNA), have become an increasingly popular tool in surveillance programs that monitor for the presence of invasive species in aquatic systems. One early application of these methods in aquatic systems was surveillance for DNA of Asian carps (specifically bighead carp Hypophthalmichthys nobilis and silver carp H. molitrix) in water samples taken from the Chicago Area Waterway System. The ability to identify DNA of a species in an environmental sample presents a potentially powerful tool because these sensitive analyses can presumably detect the presence of DNA in water even when the species is not abundant or are difficult to catch or monitor with traditional gear. Prior to research presented in this report, an initial eDNA surveillance effort was completed in selected locations in the Upper Mississippi and St. Croix Rivers in 2011 after the capture of a bighead carp in the St. Croix River near Prescott, WI. Data presented in this report were developed to duplicate the 2011 monitoring results from the Upper Mississippi and St. Croix Rivers and to provide critical insight into the technique to inform future work in these locations. We specifically sought to understand the potential confounding effects of other pathways of eDNA movement (e.g., fish-eating birds, watercraft) on the variation in background DNA by collecting water samples from (1) sites within the St. Croix River and the upper Mississippi River where the DNA of silver carp was previously detected, (2) sites considered to be free of Asian carp, and (3) a site known to have a large population of Asian carp. We also sought to establish a baseline Asian carp eDNA signature to which future eDNA sampling efforts could be compared. All samples taken as part of this effort were processed using conventional polymerase chain reaction (PCR) according to procedures outlined in the U.S. Army Corps of Engineers Quality Assurance Project Plan with minor deviations designed to enhance the rigor of our data. Presence of DNA in PCR-positive samples was confirmed by Sanger sequencing (forward and reverse) and sequences were considered positive only if sequences (forward and reverse) of ?150 base pairs had a match of ?95% to those of published sequences for bighead carp or silver carp. The DNA of bighead carp and silver carp was not detected in environmental samples collected above and below St. Croix Falls Dam on the St. Croix River, above and below the Coon Rapids Dam and below Lock and Dam 1 on the Upper Mississippi River, and from two negative control lakes, Square Lake and Lake Riley. The DNA of silver carp was detected in environmental samples collected below Lock and Dam 19 at Keokuk, Iowa, a reach of the river with high silver carp abundance. The portion (68%) of environmental samples taken below Lock and Dam 19 that were determined to contain the DNA of silver carp was similar to that reported in the scientific literature for other abundant species. The DNA of bighead carp, however, was not detected in environmental samples collected below Lock and Dam 19, a reach of the river known to have bighead carp. Previous reported detections of the DNA of silver carp in samples collected in 2011 were not replicated in this study. Additional analyses are planned for the DNA extracted from the samples collected in 2012. Those analyses may provide additional information regarding the lack of amplification of bighead carp DNA and the lengths of the sequences of silver carp DNA present in samples taken below Lock and Dam 19. These additional analyses may help inform the use of eDNA monitoring in large, complex systems like the Mississippi River.

Amberg, Jon J.; McCalla, S. Grace; Miller, Loren; Sorensen, Peter; Gaikowski, Mark P.

2013-01-01

300

Application of a high surface area solid-phase microextraction air sampling device: collection and analysis of chemical warfare agent surrogate and degradation compounds.  

PubMed

This work examines a recently improved, dynamic air sampling technique, high surface area solid-phase microextraction (HSA-SPME), developed for time-critical, high-volume sampling and analysis scenarios. The previously reported HSA-SPME sampling device, which provides 10-fold greater surface area compared to commercially available SPME fibers, allowed for an increased analyte uptake per unit time relative to exhaustive sampling through a standard sorbent tube. This sampling device has been improved with the addition of a type-K thermocouple and a custom heater control circuit for direct heating, providing precise (relative standard deviation ?1%) temperature control of the desorption process for trapped analytes. Power requirements for the HSA-SPME desorption process were 30-fold lower than those for conventional sorbent-bed-based desorption devices, an important quality for a device that could be used for field analysis. Comparisons of the HSA-SPME device when using fixed sampling times for the chemical warfare agent (CWA) surrogate compound, diisopropyl methylphosphonate (DIMP), demonstrated that the HSA-SPME device yielded a greater chromatographic response (up to 50%) relative to a sorbent-bed method. Another HSA-SPME air sampling approach, in which two devices are joined in tandem, was also evaluated for very rapid, low-level, and representative analysis when using discrete sampling times for the compounds of interest. The results indicated that subparts per billion by volume concentration levels of DIMP were detectable with short sampling times (?15 s). Finally, the tandem HSA-SPME device was employed for the headspace sampling of a CWA degradation compound, 2-(diisopropylaminoethyl) ethyl sulfide, present on cloth material, which demonstrated the capability to detect trace amounts of a CWA degradation product that is estimated to be less volatile than sarin. The rapid and highly sensitive detection features of this device may be beneficial in decision making for law enforcement, military, and civilian emergency organizations and responders, providing critical information in a contaminated environment scenario when time is of the essence. PMID:23902152

Stevens, Michael E; Tipple, Christopher A; Smith, Philip A; Cho, David S; Mustacich, Robert V; Eckenrode, Brian A

2013-09-17

301

Competitive Metagenomic DNA Hybridization Identifies Host-Specific Microbial Genetic Markers in Cow Fecal Samples  

PubMed Central

Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host-specific markers. Here we describe the application of a genome fragment enrichment (GFE) method to identify host-specific genetic markers from fecal microbial community DNA. As a proof of concept, bovine fecal DNA was challenged against a porcine fecal DNA background to select for bovine-specific DNA sequences. Bioinformatic analyses of 380 bovine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode membrane-associated and secreted proteins. Oligonucleotide primers capable of annealing to select Bacteroidales-like bovine GFE sequences exhibited extremely high specificity (>99%) in PCR assays with total fecal DNAs from 279 different animal sources. These primers also demonstrated a broad distribution of corresponding genetic markers (81% positive) among 148 different bovine sources. These data demonstrate that direct metagenomic DNA analysis by the competitive solution hybridization approach described is an efficient method for identifying potentially useful fecal genetic markers and for characterizing differences between environmental microbial communities. PMID:16751515

Shanks, Orin C.; Santo Domingo, Jorge W.; Lamendella, Regina; Kelty, Catherine A.; Graham, James E.

2006-01-01

302

Mite species identification in the production of allergenic extracts for clinical use and in environmental samples by ribosomal DNA amplification.  

PubMed

The identification of allergy-causing mites is conventionally based on morphological characters. However, molecular taxonomy using ribosomal DNA (rDNA) may be particularly useful in the analysis of mite cultures and purified mite fractions in the production of allergenic extracts. Full-length internal transcribed spacers (ITS1 and ITS2) were obtained from Dermatophagoides farinae, Dermatophagoides pteronyssinus, Dermatophagoides microceras and Euroglyphus maynei (Astigmata: Pyroglyphidae), Glycyphagus domesticus and Lepidoglyphus destructor (Astigmata: Glycyphagidae), Tyrophagus fanetzhangorum, Tyrophagus putrescentiae, Tyrophagus longior, Tyrophagus neiswanderi, Acarus farris and Acarus siro (Astigmata: Acaridae), and Blomia tropicalis (Astigmata: Echymopodidae), using mite-specific primers. Polymerase chain reaction (PCR) products were digested with HpaII and RsaI restriction enzymes in order to produce species-specific PCR restricted fragment length polymorphism (RFLP) profiles. A semi-nested re-amplification step was introduced before the RFLP in order to apply the method to environmental samples. Results demonstrate that rDNA sequences can be used for the unambiguous identification of mite species. The PCR-RFLP system allows the identification of species in purified mite fractions when the availability of intact adult mite bodies for morphological identification is limited. This reliable and straightforward PCR-RFLP system and the rDNA sequences obtained can be of use in the identification of allergy-causing mite species. PMID:24617319

Beroiz, B; Couso-Ferrer, F; Ortego, F; Chamorro, M J; Arteaga, C; Lombardero, M; Castañera, P; Hernández-Crespo, P

2014-09-01

303

The forensiX Evidence Collection Tube and Its Impact on DNA Preservation and Recovery  

PubMed Central

Biological samples are vulnerable to degradation from the time they are collected until they are analysed at the laboratory. Biological contaminants, such as bacteria, fungi, and enzymes, as well as environmental factors, such as sunlight, heat, and humidity, can increase the rate of DNA degradation. Currently, DNA samples are normally dried or frozen to limit their degradation prior to their arrival at the laboratory. In this study, the effect of the sample drying rate on DNA preservation was investigated, as well as a comparison between drying and freezing methods. The drying performances of two commercially available DNA collection tools (swab and drying tube) with different drying rates were evaluated. The swabs were used to collect human saliva, placed into the drying tubes, and stored in a controlled environment at 25°C and 60% relative humidity, or frozen at ?20°C, for 2 weeks. Swabs that were stored in fast sample drying tubes yielded 95% recoverable DNA, whereas swabs stored in tubes with slower sample drying rates yielded only 12% recoverable DNA; saliva stored in a microtube at ?20°C was used as a control. Thus, DNA sampling tools that offer rapid drying can significantly improve the preservation of DNA collected on a swab, increasing the quantity of DNA available for subsequent analysis. PMID:24288659

Garvin, Alex M.

2013-01-01

304

eSensor: an electrochemical detection-based DNA microarray technology enabling sample-to-answer molecular diagnostics  

NASA Astrophysics Data System (ADS)

DNA microarrays are becoming a widespread tool used in life science and drug screening due to its many benefits of miniaturization and integration. Microarrays permit a highly multiplexed DNA analysis. Recently, the development of new detection methods and simplified methodologies has rapidly expanded the use of microarray technologies from predominantly gene expression analysis into the arena of diagnostics. Osmetech's eSensor® is an electrochemical detection platform based on a low-to- medium density DNA hybridization array on a cost-effective printed circuit board substrate. eSensor® has been cleared by FDA for Warfarin sensitivity test and Cystic Fibrosis Carrier Detection. Other genetic-based diagnostic and infectious disease detection tests are under development. The eSensor® platform eliminates the need for an expensive laser-based optical system and fluorescent reagents. It allows one to perform hybridization and detection in a single and small instrument without any fluidic processing and handling. Furthermore, the eSensor® platform is readily adaptable to on-chip sample-to-answer genetic analyses using microfluidics technology. The eSensor® platform provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus have a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Liu, Robin H.; Longiaru, Mathew

2009-05-01

305

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

PubMed

Abstract Ancient parasite eggs were recovered from environmental samples collected at a Viking-age settlement in Viborg, Denmark, dated 1018-1030 A.D. Morphological examination identified Ascaris sp., Trichuris sp. and Fasciola sp. eggs, but size and shape did not allow species identification. By carefully selecting genetic markers, PCR amplification and sequencing of ancient DNA (aDNA) isolates resulted in identification of; the human whipworm, Trichuris trichiura, using SSUrRNA sequence homology; Ascaris sp. with 100% homology to cox1 haplotype 07 and Fasciola hepatica using ITS1 sequence homology. The identification of T. trichiura eggs indicates that human fecal material is present and hence that the Ascaris sp. haplotype 07 was most likely a human variant in Viking-age Denmark. The location of the F. hepatica finding suggests that sheep or cattle are the most likely hosts. Further, we sequenced the Ascaris sp. 18S rRNA gene in recent isolates from humans and pigs of global distribution and show that this is not a suited marker for species-specific identification. Finally, we discuss ancient parasitism in Denmark and the implementation of aDNA analysis methods in paleoparasitological studies. We argue that when employing species specific identification, soil samples offers excellent opportunities for studies of human parasite infections and of human and animal interactions of the past. PMID:25357228

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

2014-10-30

306

Isolation and characterization of tannin-degrading bacteria from faecal samples of some wild ruminants in Ethiopia  

Microsoft Academic Search

The aim of this study was to isolate and characterize bacteria capable of hydrolysing tannins from the faeces of Ethiopian ruminants adapted to feed on tannin-rich leaves. Faecal samples were collected from dikdik (Madoqua guentheri), camel (Camelus dromedaries), Grant's gazelle (Gazella granti), zebra (Equus quagga) and hartebeest (Alcelaphus buselaphus). Mixed cultures of the samples were screened for their tannin-hydrolysing capacities

Eden Ephraim; Agnes Odenyo; Mogessie Ashenafi

2005-01-01

307

Smoking related carcinogen-DNA adducts in biopsy samples of human urinary bladder: Identification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl as a major adduct  

SciTech Connect

The prevalence of covalent modifications to DNA (carcinogen-DNA adducts) in 42 human urinary bladder biopsy samples was investigated by {sup 32}P-postlabeling methods, with enhancement by both nuclease P1 treatment and 1-butanol extraction. Total mean carcinogen-DNA adduct levels and the mean levels of several specific adducts were significantly elevated in DNA samples of 13 current smokers, as opposed to 9 never smokers or 20 ex-smokers (5 years abstinence). There was no significant difference between the latter two groups. Several DNA adducts enhanced by nuclease P1 treatment were chromatographically similar to putative hydrocarbon DNA adducts reported earlier for placenta and lung DNA samples obtained from cigarette smokers. Putative aromatic amine adducts were detected by 1-butanol extraction that were not present when the samples were treated with nuclease P1. One of these displayed chromatographic behavior identical to the predominant adduct induced by the human urinary bladder carcinogen, 4-aminobiphenyl, which is present in cigarette smoke. This adduct comigrated in several thin-layer chromatographic systems with a synthetic N-(deoxyguanosin-8-yl)-4-amino(2,2{prime}-{sup 3}H)biphenyl-3{prime},5{prime}-bisphosphate marker. These data reinforce an association between cigarette smoking and DNA damage and suggest a molecular basis for the initiation of human urinary bladder cancer by cigarette smoke.

Talaska, G. (National Center for Toxicological Research, Jefferson, AR (United States) Univ. of Cincinnati, OH (United States)); Al-Juburi, A.Z.S.S. (John A. McClellan Memorial Veterans Administration Hospital, Little Rock, AR (United States)); Kadlubar, F.F. (National Center for Toxicological Research, Jefferson, AR (United States))

1991-06-15

308

Rapid Detection of Candida albicans in Clinical Samples by DNA Amplification of Common Regions from C. albicans-Secreted Aspartic Proteinase Genes  

Microsoft Academic Search

Laboratory diagnosis based on genomic amplification methods such as PCR may provide an alternative and more sensitive method than conventional culture for the early detection of deep-seated candidiasis, an increas- ing cause of morbidity and mortality among immunocompromised patients. A novel method of DNA extraction from clinical samples based on treatment with proteinase K and isolation of DNA on a

M. FLAHAUT; D. SANGLARD; M. MONOD; J. BILLE; M. ROSSIER

309

Binning of shallowly sampled metagenomic sequence fragments reveals that low abundance bacteria play important roles in sulfur cycling and degradation of complex organic polymers in an acid mine drainage community  

NASA Astrophysics Data System (ADS)

Our understanding of environmental microbiology has been greatly enhanced by community genome sequencing of DNA recovered directly the environment. Community genomics provides insights into the diversity, community structure, metabolic function, and evolution of natural populations of uncultivated microbes, thereby revealing dynamics of how microorganisms interact with each other and their environment. Recent studies have demonstrated the potential for reconstructing near-complete genomes from natural environments while highlighting the challenges of analyzing community genomic sequence, especially from diverse environments. A major challenge of shotgun community genome sequencing is identification of DNA fragments from minor community members for which only low coverage of genomic sequence is present. We analyzed community genome sequence retrieved from biofilms in an acid mine drainage (AMD) system in the Richmond Mine at Iron Mountain, CA, with an emphasis on identification and assembly of DNA fragments from low-abundance community members. The Richmond mine hosts an extensive, relatively low diversity subterranean chemolithoautotrophic community that is sustained entirely by oxidative dissolution of pyrite. The activity of these microorganisms greatly accelerates the generation of AMD. Previous and ongoing work in our laboratory has focused on reconstrucing genomes of dominant community members, including several bacteria and archaea. We binned contigs from several samples (including one new sample and two that had been previously analyzed) by tetranucleotide frequency with clustering by Self-Organizing Maps (SOM). The binning, evaluated by comparison with information from the manually curated assembly of the dominant organisms, was found to be very effective: fragments were correctly assigned with 95% accuracy. Improperly assigned fragments often contained sequences that are either evolutionarily constrained (e.g. 16S rRNA genes) or mobile elements that are not expected to reflect the tetranucleotide frequency signature of the host genome. Four unknown tetranucleotide frequency clusters with significant sequence (6 Mb total) were noted and analyzed further. Based on phylogenetic markers and BLAST results, these clusters represent low abundance bacteria including Acintobacteria, Firmicutes, and Proteobacteria. Functional analysis of these clusters revealved that the low- abundance bacteria harbor genes that could potentially encode important ecosystem functions such as sulfur utilization (e.g. polysulfide reductase) and polymer degradation (e.g. chitinase and glycoside hydrolase). We conclude that ESOM clustering of tetranucleotide frequency patterns is an effective method for rapidly binning shotgun community genomic sequences and a valuable tool for analyzing minor community members, which despite their low abundance may play crucial ecological roles.

Dick, G. J.; Andersson, A.; Banfield, J. F.

2007-12-01

310

Current Practices for DNA Sample Collection and Storage in the Pharmaceutical Industry, and Potential Areas for Harmonization: Perspective of the I-PWG  

Microsoft Academic Search

Collection and storage of DNA samples in clinical drug development programs are an important investment for the pharmaceutical industry to allow efficient evaluation of observed variability in drug response. To enable collection and future use of samples, individual companies must define (i) processes to collect specimens worldwide, (ii) whether collection is optional or mandatory, (iii) conditions and duration of sample

M A Franc; A W Warner; N Cohen; P M Shaw; P Groenen; A Snapir

2011-01-01

311

Automated sample preparation with extraction columns followed by liquid chromatography-ionspray mass spectrometry interferences, determination and degradation of polar organophosphorus pesticides in water samples  

Microsoft Academic Search

The determination of polar and\\/or thermally labile organophosphorus pesticides trichlorfon, dichlorvos, dimethoate, oxydemeton-methyl, mevinphos (cis and trans), demeton-S-methyl, fenamiphos, fenitrothion, fenthion and diazinon in water samples was investigated using solid-phase extraction followed by liquid chromatography-ionspray mass spectrometry (LC-ISP-MS). Pesticides were spiked at 0.2 ?g\\/l in ground water samples and 200 ml were preconcentrated by using an ASPEC XL system. To

C Molina; P Grasso; E Benfenati; D Barceló

1996-01-01

312

Comparison of Methods for DNA Isolation from Food Samples for Detection of Shiga Toxin-Producing Escherichia coli by Real-Time PCR  

Microsoft Academic Search

In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly. Enteric pathogens are classic potential agents of bioterror- ism. For example, Salmonella enterica serovar Typhimurium was

Loree C. Heller; Carisa R. Davis; K. Kealy Peak; David Wingfield; Andrew C. Cannons; Philip T. Amuso; Jacqueline Cattani

2003-01-01

313

Acceptability of self-collection sampling for HPV-DNA testing in low-resource settings: a mixed methods approach  

PubMed Central

Background Vaginal self-sampling with HPV-DNA tests is a promising primary screening method for cervical cancer. However, women’s experiences, concerns and the acceptability of such tests in low-resource settings remain unknown. Methods In India, Nicaragua, and Uganda, a mixed-method design was used to collect data from surveys (N?=?3,863), qualitative interviews (N?=?72; 20 providers and 52 women) and focus groups (N?=?30 women) on women’s and providers’ experiences with self-sampling, women’s opinions of sampling at home, and their future needs. Results Among surveyed women, 90% provided a self- collected sample. Of these, 75% reported it was easy, although 52% were initially concerned about hurting themselves and 24% were worried about not getting a good sample. Most surveyed women preferred self-sampling (78%). However it was not clear if they responded to the privacy of self-sampling or the convenience of avoiding a pelvic examination, or both. In follow-up interviews, most women reported that they didn’t mind self-sampling, but many preferred to have a provider collect the vaginal sample. Most women also preferred clinic-based screening (as opposed to home-based self-sampling), because the sample could be collected by a provider, women could receive treatment if needed, and the clinic was sanitary and provided privacy. Self-sampling acceptability was higher when providers prepared women through education, allowed women to examine the collection brush, and were present during the self-collection process. Among survey respondents, aids that would facilitate self-sampling in the future were: staff help (53%), additional images in the illustrated instructions (31%), and a chance to practice beforehand with a doll/model (26%). Conclusion Self-and vaginal-sampling are widely acceptable among women in low-resource settings. Providers have a unique opportunity to educate and prepare women for self-sampling and be flexible in accommodating women’s preference for self-sampling. PMID:24927941

2014-01-01

314

Quantitation of the Population Size and Metabolic Activity of a Resin Acid Degrading Bacterium in Activated Sludge Using Slot-Blot Hybridization to Measure the rRNA:rDNA Ratio.  

PubMed

The 16S rRNA:rDNA ratio is a useful parameter for measuring metabolic activity of a selected member of a complex microbial community, as in pulp effluent activated sludge systems. The RNA:DNA ratio of Sphingomonas sp. DhA-33, previously isolated from a sequencing batch reactor treating pulp mill effluent, is positively correlated with its growth rate (µ) under steady-state conditions. DhA-33 was grown in a chemostat with growth rates ranging from 0.04 to 0.15 cell divisions per hour. DhA-33 was also able to degrade dehydroabietic acid in bleached kraft mill effluent (BKME) plus mineral medium in batch culture. Slot-blot hybridization with radioactively labeled species-specific oligonucleotide probes for 16S rRNA and 16S rDNA was used to measure rRNA, rDNA, and the RNA:DNA ratio of this strain when in a mixed sludge community. An increase in DhA-33 rDNA indicated growth of DhA-33 within the community. The RNA:DNA ratio of DhA-33 increased sharply during exponential growth and declined as cells entered stationary phase. The RNA:DNA ratio decreased earlier and faster in DhA- 33/sludge co-cultures than in DhA-33 pure cultures, presumably due to an earlier depletion of nutrients. The species-specific quantification of the RNA:DNA ratio makes it possible to estimate the metabolic activity of selected members of a microbial community in situ. PMID:10758181

Muttray; Mohn

1999-11-01

315

Microbial Degradation of Octamethylcyclotetrasiloxane  

PubMed Central

The microbial degradation of low-molecular-weight polydimethylsiloxanes was investigated through laboratory experiments. Octamethylcyclotetrasiloxane was found to be biodegraded under anaerobic conditions in composted sewage sludge, as monitored by the occurrence of the main polydimethylsiloxane degradation product, dimethylsilanediol, compared to that found in experiments with sterilized control samples. PMID:10224038

Grümping, R.; Michalke, K.; Hirner, A. V.; Hensel, R.

1999-01-01

316

Forensic DNA typing of human nails at various stages of decomposition  

Microsoft Academic Search

Forensic scientists often face the problem of extracting and typing human DNA from degraded materials such as muscle and bones from decomposed bodies. Bone samples are particularly difficult and time consuming to be analyzed and other body tissues suffer from rapid deterioration. Nails are a well-known source of DNA and their composition makes them less predisposed to decomposition compared to

A. Piccinini; N. Cucurachi; F. Betti; M. Capra; S. Coco; F. D'Avila; R. Lorenzoni; A. Lovisolo

2006-01-01

317

Isolation and Characterization of Diuron-degrading Bacteria from Lotic Surface Water  

Microsoft Academic Search

The bacterial community structure of a diuron-degrading enrichment culture from lotic surface water samples was analyzed and\\u000a the diuron-degrading strains were selected using a series of techniques combining temporal temperature gradient gel electrophoresis\\u000a (TTGE) of 16 S rDNA gene V1–V3 variable regions, isolation of strains on agar plates, colony hybridization methods, and biodegradation\\u000a assays. The TTGE fingerprints revealed that diuron had

Isabelle Batisson; Stéphane Pesce; Pascale Besse-Hoggan; Martine Sancelme; Jacques Bohatier

2007-01-01

318

Degradation of phenanthrene by bacterial strain isolated from soil in oil refinery fields in Shanghai China  

Microsoft Academic Search

A bacterial strain Pseudomonas stutzeri ZP2 was identified with phenanthrene-degrading ability based on Gram staining, oxydase reaction, biochemical tests, FAME analysis, G+C content and 16S rDNA gene sequence analysis. It is the first time that P. stutzeri is reported to process the capability for phenanthrene degradation. The strain was isolated from soil samples contaminated with polycyclic aromatic hydrocarbon (PAH)-containing waste

He-Ping Zhao; Qing-Sheng Wu; Lei Wang; Xue-Tao Zhao; Hong-Wen Gao

2009-01-01

319

5-color multiplexed microwave-accelerated metal-enhanced fluorescence: detection and analysis of multiple DNA sequences from within one sample well within a few seconds.  

PubMed

We present a potentially highly sensitive and selective bio-assay for the potential detection of any five different DNA sequences from one sample in one well. The assay is based on a DNA "rapid catch and signal" (DNA-RCS) technology developed for the detection of different DNA sequences from a sample well area. Our signal amplification utilizes the metal-enhanced fluorescence (MEF) of dyes attached to the probe-DNAs, which hybridizes with the pre-formed mixture of anchor-DNA scaffolds on silver island films (SiFs). Low-power microwave irradiation accelerates both the formation of the anchor-DNA scaffold on the SiF-surface and anchor/probe DNA hybridization, i.e. "rapid catch" of target DNAs from a bulk solution, decreasing the assay run time from hours to only a few seconds. Localization of signaling dye-labels close to the SiFs make them extremely photostable, which allows for collecting/integrating the signal over a long time period. To demonstrate a 5 color DNA assay (5-plex) we have used a range of readily available Alexa™ dyes. Advantages and perspectives of the RCS-technologies ability to detect 5 different DNA sequences from within one plate-well are discussed. PMID:25263097

Dragan, Anatoliy; Geddes, Chris D

2014-11-01

320

DNA extraction for short tandem repeat typing from mixed samples using anti-human leukocyte CD45 and ABO blood group antibodies.  

PubMed

DNA testing from mixed cell samples can be difficult to use successfully in criminal investigations. Here, we present a method for the extraction of DNA from mixed bloodstains involving plural contributors, after antibody-microbead captured cell separation. This method, together with the multiplex short tandem repeat typing presented, has proven highly successful in the recovery of DNA profiles corresponding to the ABO blood type. Methodological steps include magnetic separation using leukocyte specific CD45 antibody-coated microbeads and centrifugal separation of leukocyte agglutination by ABO antibody. The detection results of variable mixed ratio showed that the target DNA was detected accurately as low as 1:512 mixed ratio, regardless of the large amount of the background DNA present. The method presented here is applicable to PCR-based identification for various kinds of mixed samples. PMID:24680125

Yano, Shizue; Honda, Katsuya; Kaminiwa, Junko; Nishi, Takeki; Iwabuchi, Yayoi; Sugano, Yukiko; Kurosu, Akira; Suzuki, Yasuhito

2014-05-01

321

Deep UV generation and direct DNA photo-interaction by harmonic nanoparticles in labelled samples  

NASA Astrophysics Data System (ADS)

A biophotonics approach based on the nonlinear optical process of second harmonic generation is presented and demonstrated on malignant human cell lines labelled by harmonic nanoparticles. The method enables independent imaging and therapeutic action, selecting each modality by simply tuning the excitation laser wavelength from infrared to visible. In particular, the generation of deep ultraviolet radiation at 270 nm allows direct interaction with nuclear DNA in the absence of photosensitizing molecules.

Staedler, Davide; Magouroux, Thibaud; Passemard, Solène; Schwung, Sebastian; Dubled, Marc; Schneiter, Guillaume Stéphane; Rytz, Daniel; Gerber-Lemaire, Sandrine; Bonacina, Luigi; Wolf, Jean-Pierre

2014-02-01

322

Dispersive derivatization liquid–liquid extraction of degradation products\\/precursors of mustards and V-agents from aqueous samples  

Microsoft Academic Search

A new derivatization and extraction technique termed as dispersive derivatization liquid–liquid extraction (DDLLE) speeds up the analysis process by removing the requirement for drying of the sample. The derivatization process takes place at the interface between the analyte containing aqueous phase and derivatization agent laden organic phase. The organic phase is highly dispersed using disperser solvent so that the total

Meehir Palit; Gary Mallard

2011-01-01

323

Detection of african Swine Fever virus DNA in blood samples stored on FTA cards from asymptomatic pigs in mbeya region, Tanzania.  

PubMed

The aim of the study was to assess whether blood samples collected onto FTA(®) cards could be used in combination with real-time PCR for the detection of African swine fever virus (ASFV) DNA in samples from resource-poor settings under the assumption that asymptomatically (sub-clinically) infected pigs may be present. Blood samples were collected from clinically healthy pigs from Mbeya Region, Tanzania. The blood samples were stored on FTA(®) cards and analysed by real-time PCR assays in duplicate; three pigs had high levels of viral DNA (Ct values of 27-29), and three pigs had a low level of viral DNA (Ct 36-45). Four pigs were positive in one of the duplicate samples only, but clear products of the expected size were obtained when the reactions were analysed by gel electrophoresis. For comparison, blood samples from pigs experimentally infected with either a pathogenic (OURT T88/1) or a non-pathogenic (OURT T88/3) isolate of ASFV were collected, stored on FTA(®) cards and analysed in the same way. The blood from pigs infected with the OURT T88/1 isolate showed high levels of viral DNA (Ct 22-33), whereas infection with non-pathogenic OURT T88/3 isolate resulted in only low levels of viral DNA (Ct 39) in samples collected at 10-14 days after inoculation. PMID:23472656

Braae, U C; Johansen, M V; Ngowi, H A; Rasmussen, T B; Nielsen, J; Uttenthal, Å

2015-02-01

324

[Isolation and characterazation of a carbazole-degrading bacterial strain].  

PubMed

A bacterial strain was isolated from soil samples using plate screening techniques. Results indicated this isolated were able to use carbazole as sole source of carbon and energy, simultaneously, including N-Methylcarbazole, 4-Hydroxycarbazole and 2,2'-Biphenol. It was identified as Flavobacterium sp. according to its morphology, and biochemical properties, and 16S rDNA sequence analysis. Utilization of carbazole by the isolates was confirmed by the increase in bacterial biomass and the decrease in substrate concentration in liquid cultures. The optimal pH and temperature for cell growth and carbazole degradation were 7.5 and 30 degrees C, respectively. Resting cells grown in Luria broth also showed activity for decomposing other heterocyclic compounds. In addition, biodegradation of carbazole was carried out with carbazole degrading strain KH-6. The results indicated that 90% of the carbazole could be degraded in the sterilized soil. And strain KH-6 could enhance the degradations of carbazole significantly. PMID:21072941

Zhang, Xiao-Fan; He, Yi-Liang

2010-09-01

325

Development of a Loop-Mediated Isothermal Amplification Method for Detection of Histoplasma capsulatum DNA in Clinical Samples  

PubMed Central

Improved methods for the detection of Histoplasma capsulatum are needed in regions with limited resources in which the organism is endemic, where delayed diagnosis of progressive disseminated histoplasmosis (PDH) results in high mortality rates. We have investigated the use of a loop-mediated isothermal amplification (LAMP) assay to facilitate rapid inexpensive molecular diagnosis of this disease. Primers for LAMP were designed to amplify the Hcp100 locus of H. capsulatum. The sensitivity and limit of detection were evaluated using DNA extracted from 91 clinical isolates of known geographic subspecies, while the assay specificity was determined using DNA extracted from 50 other fungi and Mycobacterium tuberculosis. Urine specimens (n = 6) collected from HIV-positive individuals with culture- and antigen-proven histoplasmosis were evaluated using the LAMP assay. Specimens from healthy persons (n = 10) without evidence of histoplasmosis were used as assay controls. The Hcp100 LAMP assay was 100% sensitive and specific when tested with DNA extracted from culture isolates. The median limit of detection was ?6 genomes (range, 1 to 300 genomes) for all except one geographic subspecies. The LAMP assay detected Hcp100 in 67% of antigen-positive urine specimens (4/6 specimens), and results were negative for Hcp100 in all healthy control urine specimens. We have shown that the Hcp100 LAMP assay is a rapid affordable assay that can be used to expedite culture confirmation of H. capsulatum in regions in which PDH is endemic. Further, our results indicate proof of the concept that the assay can be used to detect Histoplasma DNA in urine. Further evaluation of this assay using body fluid samples from a larger patient population is warranted. PMID:24478477

Zhou, Yitian; Theodoro, Raquel C.; Abrams, Bethany; Balajee, S. Arunmozhi; Litvintseva, Anastasia P.

2014-01-01

326

Organochlorinated pesticide degrading microorganisms isolated from contaminated soil.  

PubMed

Degradation of selected organochlorinated pesticides (?-hexachlorocyclohexane - ?-HCH, dichlorodiphenyltrichloroethane - DDT, hexachlorobenzene - HCB) by soil microorganisms was studied. Bacterial strains isolated from contaminated soil from Klatovy-Luby, Hajek and Neratovice, Czech Republic, capable of growth on the selected pesticides were isolated and characterised. These isolates were subjected to characterisation and identification by MS MALDI-TOF of whole cells and sequence analysis of 16S rRNA genes. The isolates were screened by gas chromatography for their ability to degrade the selected pesticides. Some isolates were able to degrade pesticides, and the formation of degradation products (?-pentachlorocyclohexane (?-PCCH), dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD)) observed in liquid culture confirmed their degradation capability. The isolates and DNA samples isolated from the contaminated soil were also screened for the bphA1 gene (encoding biphenyl-2,3-dioxygenase, the first enzyme in the PCB degradation pathway) and its occurrence was demonstrated. The isolates were also screened for the presence of linA, encoding dehydrochlorinase, the first enzyme of the HCH degradation pathway. The linA gene could not be found in any of the tested isolates, possibly due to the high specificity of the primers used. The isolates with the most effective degradation abilities could be used for further in situ bioremediation experiments with contaminated soil. PMID:25094051

Lovecka, Petra; Pacovska, Iva; Stursa, Petr; Vrchotova, Blanka; Kochankova, Lucie; Demnerova, Katerina

2015-01-25

327

Sampling  

NSDL National Science Digital Library

This tutorial covers some of the key terms in sampling like "population" and "sampling frame," some of the statistical terms used in sampling, and the major distinction between probability and Nonprobability sampling methods.

William Trochim (Cornell University)

2006-10-20

328

Hydroxyapatite degradation and biocompatibility  

NASA Astrophysics Data System (ADS)

Hydroxyapatite (HA) is widely used as a bioactive ceramics since it forms a chemical bonding to bone. The disadvantage of this material is its poor mechanical properties. HA can be degraded in body, which is the reason for its bioactivity, but too fast degradation rate could cause negative effects, such as macrophage present, particle generation, and even implant clinical failure. HA degradation rate will be greatly changed under many conditions: purity, HA form (i.e. bulk form, porous form, coating, or HA/polymer composites), microstructure, implant site, body conditions, etc. Although much work has been done in HA properties and application areas, the HA degradation behavior and mechanism under these different conditions are still not clear. In this research, three aspects of HA degradation have been studied: (1) Two very common impurities---Tri-Calcium Phosphate (TCP) and Calcium Oxide and their influences on HA degradation in vitro and in vivo, (2) influence of HA/polymer composite form on HA degradation, (3) HA material particle generation and related mechanism. From the in vitro and in vivo tests on bulk HA disks with various Ca/P ratios, HA degradation can clearly be found. The degradation level is different in different Ca/P ratio samples as well as in different test environments. In same test environment, non-stoichiometric HA samples have higher degradation rate than stoichiometric HA. HA/PMMA composite design successfully intensifies HA degradation both in vitro and in vivo. Grain boundary damage can be found on in vivo test samples, which has not been clearly seen on bulk HA degraded surface. HA particle generation is found in in vitro and in vivo HA/PMMA composite surface and in vivo bulk HA surface. Sintering temperature and time does affect HA grain size, and this affect HA degradation rate. Intergranular fracture is found in a several micron zone close to the Ca/P ratio 1.62 and 1.67 sample degraded surfaces. At Ca/P ratio greater than 1.667, after HA degradation in water, solution pH increases because of CaO presence.

Wang, Haibo

329

On-line solid phase extraction-liquid chromatography-mass spectrometry for trace determination of nerve agent degradation products in water samples.  

PubMed

Three primary nerve agent degradation products (ethyl-, isopropyl- and pinacolyl methylphosphonic acid) have been determined in water samples using on-line solid phase extraction-liquid chromatography and mass spectrometry (SPE-LC-MS) with electrospray ionisation. Porous graphitic carbon was employed for analyte enrichment followed by hydrophilic interaction chromatography. Diethylphosphate was applied as internal standard for quantitative determination of the alkyl methylphosphonic acids (AMPAs). By treating the samples with strong cation-exhange columns on Ba, Ag and H form, the major inorganic anions in water were removed by precipitation prior to the SPE-LC-MS determination. The AMPAs could be determined in tap water with limits of detection of 0.01-0.07 ?g L(-1) with the [M-H](-) ions extracted at an accuracy of ±5 mDa. The within and between assay precisions at analyte concentrations of 5 ?g L(-1) were 2-3%, and 5-9% relative standard deviation, respectively. The developed method was employed for determination of the AMPAs in three natural waters and a simulated waste water sample, spiked at 5 ?g L(-1). Recoveries of ethyl-, isopropyl- and pinacolyl methylphosphonic acid were 80-91%, 92-103% and 99-106%, respectively, proving the applicability of the technique for natural waters of various origins. PMID:23312321

Røen, Bent T; Sellevåg, Stig R; Lundanes, Elsa

2013-01-25

330

Detection of Kudoa septempunctata 18S ribosomal DNA in patient fecal samples from novel food-borne outbreaks caused by consumption of raw olive flounder (Paralichthys olivaceus).  

PubMed

Kudoa septempunctata is a newly identified myxosporean parasite of olive flounder (Paralichthys olivaceus) and a suspected causative agent of several food-borne gastroenteritis outbreaks in Japan. Here, we report the detection of K. septempunctata 18S ribosomal DNA in fecal samples of outbreak patients using an efficient method based on real-time PCR. We first performed a spiking experiment to assess whether our previously developed real-time PCR assay was applicable to detect K. septempunctata in feces. Simultaneously, we compared the relative extraction efficacy of K. septempunctata DNA using three commercial kits. Finally, our detection method was validated by testing 45 clinical samples obtained from 13 food-borne outbreaks associated with the consumption of raw flounder and 41 fecal samples from diarrhea patients epidemiologically unrelated to the ingestion of raw fish. We found that the FastDNA Spin Kit for Soil (MP Biomedicals) was the most efficient method for extracting K. septempunctata DNA from fecal samples. Using this kit, the detection limit of our real-time PCR assay was 1.6 × 10(1) spores per g of feces, and positive results were obtained for 21 fecal and 2 vomitus samples obtained from the food-borne outbreaks. To our knowledge, this is the first report to describe the detection of K. septempunctata DNA in patient fecal samples. We anticipate that our detection method will be useful for confirming food-borne diseases caused by K. septempunctata in laboratory investigations. PMID:22760033

Harada, Tetsuya; Kawai, Takao; Jinnai, Michio; Ohnishi, Takahiro; Sugita-Konishi, Yoshiko; Kumeda, Yuko

2012-09-01

331

Detection of Kudoa septempunctata 18S Ribosomal DNA in Patient Fecal Samples from Novel Food-Borne Outbreaks Caused by Consumption of Raw Olive Flounder (Paralichthys olivaceus)  

PubMed Central

Kudoa septempunctata is a newly identified myxosporean parasite of olive flounder (Paralichthys olivaceus) and a suspected causative agent of several food-borne gastroenteritis outbreaks in Japan. Here, we report the detection of K. septempunctata 18S ribosomal DNA in fecal samples of outbreak patients using an efficient method based on real-time PCR. We first performed a spiking experiment to assess whether our previously developed real-time PCR assay was applicable to detect K. septempunctata in feces. Simultaneously, we compared the relative extraction efficacy of K. septempunctata DNA using three commercial kits. Finally, our detection method was validated by testing 45 clinical samples obtained from 13 food-borne outbreaks associated with the consumption of raw flounder and 41 fecal samples from diarrhea patients epidemiologically unrelated to the ingestion of raw fish. We found that the FastDNA Spin Kit for Soil (MP Biomedicals) was the most efficient method for extracting K. septempunctata DNA from fecal samples. Using this kit, the detection limit of our real-time PCR assay was 1.6 × 101 spores per g of feces, and positive results were obtained for 21 fecal and 2 vomitus samples obtained from the food-borne outbreaks. To our knowledge, this is the first report to describe the detection of K. septempunctata DNA in patient fecal samples. We anticipate that our detection method will be useful for confirming food-borne diseases caused by K. septempunctata in laboratory investigations. PMID:22760033

Kawai, Takao; Jinnai, Michio; Ohnishi, Takahiro; Sugita-Konishi, Yoshiko; Kumeda, Yuko

2012-01-01

332

Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples  

Microsoft Academic Search

Forensic scientists are constantly searching for better, faster, and less expensive ways to increase the first-pass success rate of forensic sample analysis. Technological advances continue to increase the sensitivity of analysis methods to enable genotyping of samples containing minimal amounts of DNA, yet few tools are available that can simultaneously alert the analyst to both the presence of inhibition and

Katie L. Swango; William R. Hudlow; Mark D. Timken; Martin R. Buoncristiani

2007-01-01

333

Protocols for dry DNA storage and shipment at room temperature.  

PubMed

The globalization of DNA barcoding will require core analytical facilities to develop cost-effective, efficient protocols for the shipment and archival storage of DNA extracts and PCR products. We evaluated three dry-state DNA stabilization systems: commercial Biomatrica(®) DNAstable(®) plates, home-made trehalose and polyvinyl alcohol (PVA) plates on 96-well panels of insect DNA stored at 56 °C and at room temperature. Controls included unprotected samples that were stored dry at room temperature and at 56 °C, and diluted samples held at 4 °C and at -20 °C. PCR and selective sequencing were performed over a 4-year interval to test the condition of DNA extracts. Biomatrica(®) provided better protection of DNA at 56 °C and at room temperature than trehalose and PVA, especially for diluted samples. PVA was the second best protectant after Biomatrica(®) at room temperature, whereas trehalose was the second best protectant at 56 °C. In spite of lower PCR success, the DNA stored at -20 °C yielded longer sequence reads and stronger signal, indicating that temperature is a crucial factor for DNA quality which has to be considered especially for long-term storage. Although it is premature to advocate a transition to DNA storage at room temperature, dry storage provides an additional layer of security for frozen samples, protecting them from degradation in the event of freezer failure. All three forms of DNA preservation enable shipment of dry DNA and PCR products between barcoding facilities. PMID:23789643

Ivanova, Natalia V; Kuzmina, Masha L

2013-09-01

334

Protocols for dry DNA storage and shipment at room temperature  

PubMed Central

The globalization of DNA barcoding will require core analytical facilities to develop cost-effective, efficient protocols for the shipment and archival storage of DNA extracts and PCR products. We evaluated three dry-state DNA stabilization systems: commercial Biomatrica® DNAstable® plates, home-made trehalose and polyvinyl alcohol (PVA) plates on 96-well panels of insect DNA stored at 56?°C and at room temperature. Controls included unprotected samples that were stored dry at room temperature and at 56?°C, and diluted samples held at 4?°C and at ?20?°C. PCR and selective sequencing were performed over a 4-year interval to test the condition of DNA extracts. Biomatrica® provided better protection of DNA at 56?°C and at room temperature than trehalose and PVA, especially for diluted samples. PVA was the second best protectant after Biomatrica® at room temperature, whereas trehalose was the second best protectant at 56?°C. In spite of lower PCR success, the DNA stored at ?20?°C yielded longer sequence reads and stronger signal, indicating that temperature is a crucial factor for DNA quality which has to be considered especially for long-term storage. Although it is premature to advocate a transition to DNA storage at room temperature, dry storage provides an additional layer of security for frozen samples, protecting them from degradation in the event of freezer failure. All three forms of DNA preservation enable shipment of dry DNA and PCR products between barcoding facilities. PMID:23789643

Ivanova, Natalia V; Kuzmina, Masha L

2013-01-01

335

DNA barcode based wildlife forensics for resolving the origin of claw samples using a novel primer cocktail.  

PubMed

Abstract Excessive wildlife hunting for commercial purposes can have negative impacts on biodiversity and may result in species extinction. To ensure compliance with legal statutes, forensic identification approaches relying on molecular markers may be used to identify the species of origin of animal material from hairs, claw, blood, bone, or meat. Using this approach, DNA sequences from the COI "barcoding" gene have been used to identify material from a number of domesticated animal species. However, many wild species of carnivores still present great challenges in generating COI barcodes using standard "universal" primer pairs. In the work presented here, the mitochondrial COI gene was successfully amplified using a novel primer cocktail, and the products were sequenced to determine the species of twenty one unknown samples of claw material collected as part of forensic wildlife case investigations. Sixteen of the unknown samples were recognized to have originated from either Panthera leo or P. pardus individuals. The remaining five samples could be identified only to the family level due to the absence of reference animal sequences. This is the first report on the use of COI sequences for the identification of P. pardus and P. leo from claw samples as part of forensic investigations in India. The study also highlights the need for adequate reference material to aid in the resolution of suspected cases of illegal wildlife harvesting. PMID:25492536

Khedkar, Gulab D; Abhayankar, Shil Bapurao; Nalage, Dinesh; Ahmed, Shaikh Nadeem; Khedkar, Chandraprakash D

2014-12-10

336

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

PubMed

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

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

2014-02-11

337

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

PubMed Central

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

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

2014-01-01

338

DNA Detectives  

NSDL National Science Digital Library

Many of the revolutionary changes that have occurred in biology since 1970 can be attributed directly to the ability to manipulate DNA in defined ways. The principal tools for this recombinant DNA technology are enzymes that can "cut and "paste" DNA. Restriction enzymes are the "chemical scissors" of the molecular biologist; these enzymes cut DNA at specific nucleotide sequences. A sample of someone's DNA, incubated with restriction enzymes, is reduced to millions of DNA fragments of varying sizes. A DNA sample from a different person would have a different nucleotide sequence and would thus be enzymatically "chopped up" into a very different collection of fragments. We have been asked to apply DNA fingerprinting to determine which suspect should be charged with a crime perpetrated in our city.

BEGIN:VCARD VERSION:2.1 FN:Suzanne Black N:Black;Suzanne ORG:Inglemoor High School REV:2005-04-09 END:VCARD

1995-06-30

339

The half-life of DNA in bone: measuring decay kinetics in 158 dated fossils  

PubMed Central

Claims of extreme survival of DNA have emphasized the need for reliable models of DNA degradation through time. By analysing mitochondrial DNA (mtDNA) from 158 radiocarbon-dated bones of the extinct New Zealand moa, we confirm empirically a long-hypothesized exponential decay relationship. The average DNA half-life within this geographically constrained fossil assemblage was estimated to be 521 years for a 242 bp mtDNA sequence, corresponding to a per nucleotide fragmentation rate (k) of 5.50 × 10–6 per year. With an effective burial temperature of 13.1°C, the rate is almost 400 times slower than predicted from published kinetic data of in vitro DNA depurination at pH 5. Although best described by an exponential model (R2 = 0.39), considerable sample-to-sample variance in DNA preservation could not be accounted for by geologic age. This variation likely derives from differences in taphonomy and bone diagenesis, which have confounded previous, less spatially constrained attempts to study DNA decay kinetics. Lastly, by calculating DNA fragmentation rates on Illumina HiSeq data, we show that nuclear DNA has degraded at least twice as fast as mtDNA. These results provide a baseline for predicting long-term DNA survival in bone. PMID:23055061

Allentoft, Morten E.; Collins, Matthew; Harker, David; Haile, James; Oskam, Charlotte L.; Hale, Marie L.; Campos, Paula F.; Samaniego, Jose A.; Gilbert, M. Thomas P.; Willerslev, Eske; Zhang, Guojie; Scofield, R. Paul; Holdaway, Richard N.; Bunce, Michael

2012-01-01

340

Bacterial diversity in water samples from uranium wastes as demonstrated by 16S rDNA and ribosomal intergenic spacer amplification retrievals.  

PubMed

Bacterial diversity was assessed in water samples collected from several uranium mining wastes in Ger many and in the United States by using 16S rDNA and ribosomal intergenic spacer amplification retrievals. The results obtained using the 16S rDNA retrieval showed that the samples collected from the uranium mill tailings of Schlema/Alberoda, Germany, were predominated by Nitrospina-like bacteria, whereas those from the mill tailings of Shiprock, New Mexico, USA, were predominated by gamma-Pseudomonas and Frauteria spp. Additional smaller populations of the Cytophaga-Flavobacterium-Bacteroides group and alpha- and delta-Proteobacteria were identified in the Shiprock samples as well. Proteobacteria and Cytophaga-Flavobacterium-Bacteroides were also found in the third uranium mill tailings studied, Gittersee/Coschütz, Germany, but the groups of the predominant clones were rather small. Most of the clones of the Gittersee/Coschütz samples represented individual sequences, which indicates a high level of bacterial diversity. The samples from the fourth uranium waste studied, Steinsee Deponie B1, Germany, were predominantly occupied by Acinetobacter spp. The ribosomal intergenic spacer amplification retrieval provided results complementary to those obtained by the 16S rDNA analyses. For instance, in the Shiprock samples, an additional predominant bacterial group was identified and affiliated with Nitrosomonas sp., whereas in the Gittersee/Coschütz samples, anammox populations were identified that were not retrieved by the applied 16S rDNA approach. PMID:16333330

Radeva, Galina; Selenska-Pobell, Sonja

2005-11-01

341

The stability and degradation of dietary DNA in the gastrointestinal tract of mammals: implications for horizontal gene transfer and the biosafety of GMOs.  

PubMed

The fate of dietary DNA in the gastrointestinal tract (GIT) of animals has gained renewed interest after the commercial introduction of genetically modified organisms (GMO). Among the concerns regarding GM food, are the possible consequences of horizontal gene transfer (HGT) of recombinant dietary DNA to bacteria or animal cells. The exposure of the GIT to dietary DNA is related to the extent of food processing, food composition, and to the level of intake. Animal feeding studies have demonstrated that a minor amount of fragmented dietary DNA may resist the digestive process. Mammals have been shown to take up dietary DNA from the GIT, but stable integration and expression of internalized DNA has not been demonstrated. Despite the ability of several bacterial species to acquire external DNA by natural transformation, in vivo transfer of dietary DNA to bacteria in the intestine has not been detected in the few experimental studies conducted so far. However, major methodological limitations and knowledge gaps of the mechanistic aspects of HGT calls for methodological improvements and further studies to understand the fate of various types of dietary DNA in the GIT. PMID:22059960

Rizzi, Aurora; Raddadi, Noura; Sorlini, Claudia; Nordgrd, Lise; Nielsen, Kaare Magne; Daffonchio, Daniele

2012-01-01

342

Noise sampling method: an ANOVA approach allowing robust selection of differentially regulated genes measured by DNA  

E-print Network

are those which are differentially expressed. Two common methods for gene selection are: a) selection@cs.wayne.edu. 1 Introduction Many microarray experiments aim at comparing gene expression levels in two different is the identification of the genes that are differentially expressed between the two samples. Although simple

Draghici, Sorin

343

Collecting, archiving and processing DNA from wildlife samples using FTA® databasing paper  

Microsoft Academic Search

BACKGROUND: Methods involving the analysis of nucleic acids have become widespread in the fields of traditional biology and ecology, however the storage and transport of samples collected in the field to the laboratory in such a manner to allow purification of intact nucleic acids can prove problematical. RESULTS: FTA® databasing paper is widely used in human forensic analysis for the

LM Smith; LA Burgoyne

2004-01-01

344

Presence of human mycoplasma DNA in gastric tissue samples from Korean chronic gastritis patients.  

PubMed

We aimed to determine whether mycoplasmas are present in Korean chronic gastritis, and to understand their roles in gastric cancer tumorigenesis, because mycoplasmas resemble Helicobacter pylori in terms of ammonia production and induction of inflammatory cytokines in immune and non-immune cells. The presence and identity of mycoplasmas were assessed by semi-nested PCR and sequencing, and the results were compared with pathologic data. Fifty-six samples collected from Korean chronic gastritis patients were used for this study. Twenty-three (41.1%) were positive for mycoplasmas. Eighteen sequenced samples contained a single human mycoplasma or two mycoplasmas, which were identified as Mycoplasma faucium (13/18), M. fermentans (3/18), M. orale (1/18), M. salivarium (2/18), and M. spermatophilum (1/18). Mycoplasma-infected chronic gastritis samples showed significantly more severe neutrophil infiltration than non-infected samples (P = 0.0135). Mycoplasma profiles in the oral cavity (M. salivarium is major) and stomach were different, and the presence of significant proinflammatory responses in mycoplasma-positive patients suggests that the mycoplasmas are not simply contaminants. Further studies are required to understand whether mycoplasmas play a role in gastric tumorigenesis. PMID:15072588

Kwon, Hyuk-Joon; Kang, Jeong-Ok; Cho, Sun-Hee; Kang, Hee-Bum; Kang, Kyung-Ah; Kim, Jeong-Ki; Kang, Yoon-Suk; Song, Byung-Cheol; Kang, Hyun-Wook; Shim, Mi-Ja; Kim, Hee-Sun; Kim, Young-Bae; Hahm, Ki-Baeg; Kim, Bum-Joon; Kook, Myeong-Cherl; Chung, Myung-Hee; Hyun, Jin-Won

2004-04-01

345

Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments  

Microsoft Academic Search

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments was used to explore the genetic diversity\\u000a of hydrothermal vent microbial communities, specifically to determine the importance of sulfur-oxidizing bacteria therein.\\u000a DGGE analysis of two different hydrothermal vent samples revealed one PCR band for one sample and three PCR bands for the\\u000a other sample, which probably correspond to the dominant

Gerard Muyzer; Andreas Teske; Carl O. Wirsen; Holger W. Jannasch

1995-01-01

346

Optimization and Comparison of ESI and APCI LC-MS/MS Methods: A Case Study of Irgarol 1051, Diuron, and their Degradation Products in Environmental Samples  

NASA Astrophysics Data System (ADS)

A systematic and detailed optimization strategy for the development of atmospheric pressure ionization (API) LC-MS/MS methods for the determination of Irgarol 1051, Diuron, and their degradation products (M1, DCPMU, DCPU, and DCA) in water, sediment, and mussel is described. Experimental design was applied for the optimization of the ion sources parameters. Comparison of ESI and APCI was performed in positive- and negative-ion mode, and the effect of the mobile phase on ionization was studied for both techniques. Special attention was drawn to the ionization of DCA, which presents particular difficulty in API techniques. Satisfactory ionization of this small molecule is achieved only with ESI positive-ion mode using acetonitrile in the mobile phase; the instrumental detection limit is 0.11 ng/mL. Signal suppression was qualitatively estimated by using purified and non-purified samples. The sample preparation for sediments and mussels is direct and simple, comprising only solvent extraction. Mean recoveries ranged from 71% to 110%, and the corresponding (%) RSDs ranged between 4.1 and 14%. The method limits of detection ranged between 0.6 and 3.5 ng/g for sediment and mussel and from 1.3 to 1.8 ng/L for sea water. The method was applied to sea water, marine sediment, and mussels, which were obtained from marinas in Attiki, Greece. Ion ratio confirmation was used for the identification of the compounds.

Maragou, Niki C.; Thomaidis, Nikolaos S.; Koupparis, Michael A.

2011-10-01

347

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

PubMed

An integrated and multiplexed on-line instrument starting from DNA templates to their primary sequences has been demonstrated based on multiplexed microfluidics and capillary array electrophoresis. The instrument automatically processes eight templates through reaction, purification, denaturation, preconcentration, injection, separation, and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were utilized to manage flow and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an eight-capillary array in a hot-air thermal cycler. Subsequently, the sequencing ladders are directly loaded into separate size exclusion chromatographic columns operated at approximately 60 degrees C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at approximately 70 degrees C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. The raw data allow base calling up to 460 bp with an accuracy of 98%. The system is scalable to a 96-capillary array and will benefit not only high-speed, high-throughput DNA sequencing but also genetic typing. PMID:9784747

Tan, H; Yeung, E S

1998-10-01

348

Comparative effect of microwaves and boiling on the denaturation of DNA  

SciTech Connect

The effect of heat and microwave denaturation of small volumes of double-stranded plasmid DNA has been compared. Samples of intact plasmid DNA had plasmid DNA linearized by digestion with EcoRI were conventionally denatured in a boiling water bath or denatured by 2450 MHz of microwave energy for 0-300 s. Heat denaturation for periods longer than 120 s caused breakdown of linearized plasmid DNA; however, microwave denaturation for 10-300 s caused no apparent degradation of linearized DNA. Breakdown of DNA forms II and III was noted in plasmid DNA subjected to 300 s of either heat or microwave denaturation but breakdown of forms II and III occurred more quickly with heat than with microwave treatment. Microwave treatment was also found to be better than heat to denature 32P-labeled DNA probes subsequently used to detect homologous DNA samples immobilized on nitrocellulose filters. A microwave-treated 32P-labeled DNA probe was able to hybridize to DNA samples 20 times more dilute than a heat-treated 32P-labeled DNA probe. Depending on the form of DNA to be analyzed, these results indicate that small volumes of DNA solutions and radiolabeled DNA probes can be effectively denatured in a conventional microwave oven.

Stroop, W.G.; Schaefer, D.C. (Veterans Administration Medical Center, Salt Lake City, UT (USA))

1989-11-01

349

Reliable and sensitive detection of fragile X (expanded) alleles in clinical prenatal DNA samples with a fast turnaround time.  

PubMed

This study evaluated a large set of blinded, previously analyzed prenatal DNA samples with a novel, CGG triplet-repeat primed (TP)-PCR assay (Amplidex FMR1 PCR Kit; Asuragen, Austin, TX). This cohort of 67 fetal DNAs contained 18 full mutations (270 to 1100 repeats, including 1 mosaic), 12 premutations (59 to 150 repeats), 9 intermediate mutations (54 to 58 repeats), and 28 normal samples (17 to 50 repeats, including 3 homozygous female samples). TP-PCR accurately identified FMR1 genotypes, ranging from normal to full- mutation alleles, with a 100% specificity (95% CI, 85.0% to 100%) and a 97.4% sensitivity (95% CI, 84.9% to 99.9%) in comparison with Southern blot analysis results. Exact sizing was possible for a spectrum of normal, intermediate, and premutation (up to 150 repeats) alleles, but CGG repeat numbers >200 are only identified as full mutations. All homozygous alleles were correctly resolved. The assay is also able to reproducibly detect a 2.5% premutation and a 3% full-mutation mosaicism in a normal male background, but a large premutation in a full male mutation background was masked when the amount of the latter was >5%. Implementation of this TP-PCR will significantly reduce reflex testing using Southern blot analyses. Additional testing with methylation-informative techniques might still be needed for a few cases with (large) premutations or full mutations. PMID:22921311

Seneca, Sara; Lissens, Willy; Endels, Kristof; Caljon, Ben; Bonduelle, Maryse; Keymolen, Kathleen; De Rademaeker, Marjan; Ullmann, Urielle; Haentjens, Patrick; Van Berkel, Kim; Van Dooren, Sonia

2012-11-01

350

Somatic rearrangements across cancer reveal classes of samples with distinct patterns of DNA breakage and rearrangement-induced hypermutability  

PubMed Central

Whole-genome sequencing using massively parallel sequencing technologies enables accurate detection of somatic rearrangements in cancer. Pinpointing large numbers of rearrangement breakpoints to base-pair resolution allows analysis of rearrangement microhomology and genomic location for every sample. Here we analyze 95 tumor genome sequences from breast, head and neck, colorectal, and prostate carcinomas, and from melanoma, multiple myeloma, and chronic lymphocytic leukemia. We discover three genomic factors that are significantly correlated with the distribution of rearrangements: replication time, transcription rate, and GC content. The correlation is complex, and different patterns are observed between tumor types, within tumor types, and even between different types of rearrangements. Mutations in the APC gene correlate with and, hence, potentially contribute to DNA breakage in late-replicating, low %GC, untranscribed regions of the genome. We show that somatic rearrangements display less microhomology than germline rearrangements, and that breakpoint loci are correlated with local hypermutability with a particular enrichment for transversions. PMID:23124520

Drier, Yotam; Lawrence, Michael S.; Carter, Scott L.; Stewart, Chip; Gabriel, Stacey B.; Lander, Eric S.; Meyerson, Matthew; Beroukhim, Rameen; Getz, Gad

2013-01-01

351

Predicting Clopidogrel Response Using DNA Samples Linked to an Electronic Health Record  

PubMed Central

Variants in ABCB1 and CYP2C19 have been identified as predictors of cardiac events during clopidogrel therapy initiated after myocardial infarction (MI) or percutaneous coronary intervention (PCI). In addition, PON1 has recently been associated with stent thrombosis. The reported effects of these variants have not yet been replicated in a real-world setting. We used BioVU, the Vanderbilt DNA repository linked to de-identified electronic health records (EHRs), to find data on patients who were on clopidogrel treatment after an MI and/or a PCI; among these, we identified those who had experienced one or more recurrent cardiac events while on treatment (cases, n = 225) and those who had not experienced any cardiac event while on treatment (controls, n = 468). We found that CYP2C19*2 (hazard ratio (HR) 1.54, 95% confidence interval (CI) 1.16–2.06, P = 0.003) and ABCB1 (hr 1.28, 95% CI 1.04–1.57, P = 0.018), but not PON1 (HR 0.91, 95% CI 0.73–1.12, P = 0.370), were associated with recurrent events. In this population, genetic signals for clopidogrel resistance in ABCB1 and CYP2C19 were replicated, supporting the use of EHRs for pharmacogenomic studies. Our data do not show an association between PON1 and recurrent cardiovascular events. PMID:22190063

Delaney, JT; Ramirez, AH; Bowton, E; Pulley, JM; Basford, MA; Schildcrout, JS; Shi, Y; Zink, R; Oetjens, M; Xu, H; Cleator, JH; Jahangir, E; Ritchie, MD; Masys, DR; Roden, DM; Crawford, DC; Denny, JC

2013-01-01

352

DNA Barcode Authentication of Wood Samples of Threatened and Commercial Timber Trees within the Tropical Dry Evergreen Forest of India  

PubMed Central

Background India is rich with biodiversity, which includes a large number of endemic, rare and threatened plant species. Previous studies have used DNA barcoding to inventory species for applications in biodiversity monitoring, conservation impact assessment, monitoring of illegal trading, authentication of traded medicinal plants etc. This is the first tropical dry evergreen forest (TDEF) barcode study in the World and the first attempt to assemble a reference barcode library for the trees of India as part of a larger project initiated by this research group. Methodology/Principal Findings We sampled 429 trees representing 143 tropical dry evergreen forest (TDEF) species, which included 16 threatened species. DNA barcoding was completed using rbcL and matK markers. The tiered approach (1st tier rbcL; 2nd tier matK) correctly identified 136 out of 143 species (95%). This high level of species resolution was largely due to the fact that the tree species were taxonomically diverse in the TDEF. Ability to resolve taxonomically diverse tree species of TDEF was comparable among the best match method, the phylogenetic method, and the characteristic attribute organization system method. Conclusions We demonstrated the utility of the TDEF reference barcode library to authenticate wood samples from timber operations in the TDEF. This pilot research study will enable more comprehensive surveys of the illegal timber trade of threatened species in the TDEF. This TDEF reference barcode library also contains trees that have medicinal properties, which could be used to monitor unsustainable and indiscriminate collection of plants from the wild for their medicinal value. PMID:25259794

Nithaniyal, Stalin; Newmaster, Steven G.; Ragupathy, Subramanyam; Krishnamoorthy, Devanathan; Vassou, Sophie Lorraine; Parani, Madasamy

2014-01-01

353

Real-time PCR detection and quantification of elephantid DNA: species identification for highly processed samples associated with the ivory trade.  

PubMed

The ivory industry is the single most serious threat to global elephant populations. A highly sensitive, species-specific real-time PCR assay has been developed to detect and quantify African elephant (Loxodonta africana), Asian elephant (Elephas maximus) and Woolly Mammoth (Mammuthus primigenius) mitochondrial DNA from highly processed samples involved in the international ivory trade. This assay is especially useful for highly processed samples where there are no distinguishing morphological features to identify the species of origin. Using species-specific Taqman(®) probes targeting a region of the mitochondrial cytochrome b gene, we developed an assay that can be used to positively identify samples containing elephant or Woolly mammoth DNA faster and more cost-effectively than traditional sequencing methods. Furthermore, this assay provides a diagnostic result based on probe hybridization that eliminates ambiguities associated with traditional DNA sequence protocols involving low template DNA. The real-time method is highly sensitive, producing accurate and reproducible results in samples with as few as 100 copies of template DNA. This protocol can be applied to the enforcement of the Convention on the International Trade of Endangered Species (CITES), when positive identification of species from illegally traded products is required by conservation officers in wildlife forensic cases. PMID:22257967

Wozney, Kristyne Michelle; Wilson, Paul J

2012-06-10

354

Degradation process of lead chromate in paintings by Vincent van Gogh studied by means of synchrotron X-ray spectromicroscopy and related methods. 1. Artificially aged model samples.  

PubMed

On several paintings by artists of the end of the 19th century and the beginning of the 20th Century a darkening of the original yellow areas, painted with the chrome yellow pigment (PbCrO(4), PbCrO(4)·xPbSO(4), or PbCrO(4)·xPbO) is observed. The most famous of these are the various Sunflowers paintings Vincent van Gogh made during his career. In the first part of this work, we attempt to elucidate the degradation process of chrome yellow by studying artificially aged model samples. In view of the very thin (1-3 ?m) alteration layers that are formed, high lateral resolution spectroscopic methods such as microscopic X-ray absorption near edge (?-XANES), X-ray fluorescence spectrometry (?-XRF), and electron energy loss spectrometry (EELS) were employed. Some of these use synchrotron radiation (SR). Additionally, microscopic SR X-ray diffraction (SR ?-XRD), ?-Raman, and mid-FTIR spectroscopy were employed to completely characterize the samples. The formation of Cr(III) compounds at the surface of the chrome yellow paint layers is particularly observed in one aged model sample taken from a historic paint tube (ca. 1914). About two-thirds of the chromium that is present at the surface has reduced from the hexavalent to the trivalent state. The EELS and ?-XANES spectra are consistent with the presence of Cr(2)O(3)·2H(2)O (viridian). Moreover, as demonstrated by ?-XANES, the presence of another Cr(III) compound, such as either Cr(2)(SO(4))(3)·H(2)O or (CH(3)CO(2))(7)Cr(3)(OH)(2) [chromium(III) acetate hydroxide], is likely. PMID:21314201

Monico, Letizia; Van der Snickt, Geert; Janssens, Koen; De Nolf, Wout; Miliani, Costanza; Verbeeck, Johan; Tian, He; Tan, Haiyan; Dik, Joris; Radepont, Marie; Cotte, Marine

2011-02-15

355

Accuracy and Cost-Effectiveness of Cervical Cancer Screening by High-Risk HPV DNA Testing of Self-Collected Vaginal Samples  

PubMed Central

Objective Estimate the accuracy and cost-effectiveness of cervical cancer screening strategies based on high-risk HPV DNA testing of self-collected vaginal samples. Materials and Methods A subset of 1,665 women (18-50 years of age) participating in a cervical cancer screening study were screened by liquid-based cytology and by high-risk HPV DNA testing of both self-collected vaginal swab samples and clinician-collected cervical samples. Women with positive/abnormal screening test results and a subset of women with negative screening test results were triaged to colposcopy. Based on individual and combined test results, five screening strategies were defined. Estimates of sensitivity and specificity for cervical intraepithelial neoplasia grade 2 or worse were calculated and a Markov model was used to estimate the incremental cost-effectiveness ratios (ICERs) for each strategy. Results Compared to cytology-based screening, high-risk HPV DNA testing of self-collected vaginal samples was more sensitive (68%, 95%CI=58%-78% versus 85%, 95%CI=76%-94%) but less specific (89%, 95%CI=86%-91% versus 73%, 95%CI=67%-79%). A strategy of high-risk HPV DNA testing of self-collected vaginal samples followed by cytology triage of HPV positive women, was comparably sensitive (75%, 95%CI=64%-86%) and specific (88%, 95%CI=85%-92%) to cytology-based screening. In-home self-collection for high-risk HPV DNA detection followed by in-clinic cytology triage had a slightly lower lifetime cost and a slightly higher quality-adjusted life expectancy than did cytology-based screening (ICER of triennial screening compared to no screening was $9,871/QALY and $12,878/QALY, respectively). Conclusions Triennial screening by high-risk HPV DNA testing of in-home, self-collected vaginal samples followed by in-clinic cytology triage was cost-effective. PMID:20592553

Balasubramanian, Akhila; Kulasingam, Shalini L.; Baer, Atar; Hughes, James P.; Myers, Evan R.; Mao, Constance; Kiviat, Nancy B.; Koutsky, Laura A.

2010-01-01

356

Loop Mediated Isothermal Amplification (LAMP) Accurately Detects Malaria DNA from Filter Paper Blood Samples of Low Density Parasitaemias  

PubMed Central

Background Loop mediated isothermal amplification (LAMP) provides an opportunity for improved, field-friendly detection of malaria infections in endemic areas. However data on the diagnostic accuracy of LAMP for active case detection, particularly low-density parasitaemias, are lacking. We therefore evaluated the performance of a new LAMP kit compared with PCR using DNA from filter paper blood spots. Methods and Findings Samples from 865 fever patients and 465 asymptomatic individuals collected in Zanzibar were analysed for Pan (all species) and Pf (P. falciparum) DNA with the Loopamp MALARIA Pan/Pf kit. Samples were amplified at 65°C for 40 minutes in a real-time turbidimeter and results were compared with nested PCR. Samples with discordant results between LAMP and nested PCR were analysed with real-time PCR. The real-time PCR corrected nested PCR result was defined as gold standard. Among the 117 (13.5%) PCR detected P. falciparum infections from fever patients (mean parasite density 7491/µL, range 6–782,400) 115, 115 and 111 were positive by Pan-LAMP, Pf-LAMP and nested PCR, respectively. The sensitivities were 98.3% (95%CI 94–99.8) for both Pan and Pf-LAMP. Among the 54 (11.6%) PCR positive samples from asymptomatic individuals (mean parasite density 10/µL, range 0–4972) Pf-LAMP had a sensitivity of 92.7% (95%CI 80.1–98.5) for detection of the 41 P. falciparum infections. Pan-LAMP had sensitivities of 97% (95%CI 84.2–99.9) and 76.9% (95%CI 46.2–95) for detection of P. falciparum and P. malariae, respectively. The specificities for both Pan and Pf-LAMP were 100% (95%CI 99.1–100) in both study groups. Conclusion Both components of the Loopamp MALARIA Pan/Pf detection kit revealed high diagnostic accuracy for parasite detection among fever patients and importantly also among asymptomatic individuals of low parasite densities from minute blood volumes preserved on filter paper. These data support LAMPs potential role for improved detection of low-density malaria infections in pre-elimination settings. PMID:25105591

González, Iveth J.; Polley, Spencer D.; Bell, David; Shakely, Delér; Msellem, Mwinyi I.; Björkman, Anders; Mårtensson, Andreas

2014-01-01

357

DNA barcoding and metabarcoding of standardized samples reveal patterns of marine benthic diversity.  

PubMed

Documenting the diversity of marine life is challenging because many species are cryptic, small, and rare, and belong to poorly known groups. New sequencing technologies, especially when combined with standardized sampling, promise to make comprehensive biodiversity assessments and monitoring feasible on a large scale. We used this approach to characterize patterns of diversity on oyster reefs across a range of geographic scales comprising a temperate location [Virginia (VA)] and a subtropical location [Florida (FL)]. Eukaryotic organisms that colonized multilayered settlement surfaces (autonomous reef monitoring structures) over a 6-mo period were identified by cytochrome c oxidase subunit I barcoding (>2-mm mobile organisms) and metabarcoding (sessile and smaller mobile organisms). In a total area of ?15.64 m(2) and volume of ?0.09 m(3), 2,179 operational taxonomic units (OTUs) were recorded from 983,056 sequences. However, only 10.9% could be matched to reference barcodes in public databases, with only 8.2% matching barcodes with both genus and species names. Taxonomic coverage was broad, particularly for animals (22 phyla recorded), but 35.6% of OTUs detected via metabarcoding could not be confidently assigned to a taxonomic group. The smallest size fraction (500 to 106 ?m) was the most diverse (more than two-thirds of OTUs). There was little taxonomic overlap between VA and FL, and samples separated by ?2 m were significantly more similar than samples separated by ?100 m. Ground-truthing with independent assessments of taxonomic composition indicated that both presence-absence information and relative abundance information are captured by metabarcoding data, suggesting considerable potential for ecological studies and environmental monitoring. PMID:25646458

Leray, Matthieu; Knowlton, Nancy

2015-02-17

358

DNA barcoding of authentic and substitute samples of herb of the family Asparagaceae and Asclepiadaceae based on the ITS2 region  

PubMed Central

Background: Herbal drugs used to treat illness according to Ayurveda are often misidentified or adulterated with similar plant materials. Objective: To aid taxonomical identification, we used DNA barcoding to evaluate authentic and substitute samples of herb and phylogenetic relationship of four medicinal plants of family Asparagaceace and Asclepiadaceae. Materials and Methods: DNA extracted from dry root samples of two authentic and two substitutes of four specimens belonging to four species were subjected to polymerase chain reaction (PCR) and DNA sequencing. Primers for nuclear DNA (nu ITS2) and plastid DNA (matK and rpoC1) were used for PCR and sequence analysis was performed by Clustal W. The intraspecific variation and interspecific divergence were calculated using MEGA V 4.0. Statistical Analysis: Kimura's two parameter model, neighbor joining and bootstrapping methods were used in this work. Results: The result indicates the efficiency of amplification for ITS2 candidate DNA barcodes was 100% for four species tested. The average interspecific divergence is 0.12 and intraspecific variation was 0.232 in the case of two Asparagaceae species. In two Asclepiadaceae species, average interspecific divergence and intraspecific variation were 0.178 and 0.004 respectively. Conclusions: Our findings show that the ITS2 region can effectively discriminate Asparagus racemosus and Hemidesmus indicus from its substitute samples and hence can resolve species admixtures in raw samples. The ITS2 region may be used as one of the standard DNA barcodes to identify closely related species of family Asclepiadaceae but was noninformative for Asparagaceae species suggesting a need for the development of new markers for each family. More detailed studies involving more species and substitutes are warranted. PMID:23125510

Rai, Padmalatha S; Bellampalli, Ravishankara; Dobriyal, Rajendra M; Agarwal, Amit; Satyamoorthy, K; Narayana, DB Anantha

2012-01-01

359

Direct detection of Theileria annulata in bovine blood samples using standard and isothermal DNA amplification approaches.  

PubMed

Tropical theileriosis is a tick-borne disease responsible for important health problems in cattle, caused by the hemoprotozoan Theileria annulata. Traditionally, detection of Theileria pathogens in infected animals requires the microscopic examination of stained-blood smears and serological methods. Molecular diagnostic assays have been developed for the detection of Theileria parasites, including PCR-based and reverse line blotting approaches, but these methods usually demand qualified personnel, complex instrumentation, and expensive materials. Loop-mediated isothermal amplification (LAMP) can facilitate the design of molecular assays independent of the use of sophisticated equipment. In this chapter we describe the application of two molecular assays for the direct detection of T. annulata in bovine blood samples, based in real-time PCR and LAMP, both targeting the Tams1-encoding gene of this parasite. PMID:25399096

Gomes, Jacinto; Inácio, João

2015-01-01

360

Automated extraction of DNA from blood and PCR setup using a Tecan Freedom EVO liquid handler for forensic genetic STR typing of reference samples.  

PubMed

We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO liquid handler mounted with the Te-MagS magnetic separation device (Tecan, Männedorf, Switzerland). The protocols were validated for accredited forensic genetic work according to ISO 17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained the samples were washed with MilliQ water before the return of the DNA extracts. The PCR was setup in 96-well microtiter plates. The methods were validated for the kits: AmpF?STR Identifiler, SGM Plus and Yfiler (Applied Biosystems, Foster City, CA), GenePrint FFFL and PowerPlex Y (Promega, Madison, WI). The automated protocols allowed for extraction and addition of PCR master mix of 96 samples within 3.5h. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic genetic short tandem repeat typing can be implemented on a simple automated liquid handler leading to the reduction of manual work, and increased quality and throughput. PMID:21609694

Stangegaard, Michael; Frøslev, Tobias G; Frank-Hansen, Rune; Hansen, Anders J; Morling, Niels

2011-04-01

361

Simultaneous determination of florfenicol with its metabolite based on modified quick, easy, cheap, effective, rugged, and safe sample pretreatment and evaluation of their degradation behavior in agricultural soils.  

PubMed

A simple and simultaneous method for the determination of florfenicol and its metabolite florfenicol amine in agricultural soils using modified quick, easy, cheap, effective, rugged, and safe sample pretreatment and reversed-phase high-performance liquid chromatography with tandem mass spectrometry is presented. Florfenicol and its metabolite florfenicol amine residues in agricultural soils were extracted with alkalized acetonitrile and an aliquot was cleaned up with Si(CH2 )3 NH (CH2 )2 NH2 and C18 sorbent, which were powder materials. High-performance liquid chromatography with tandem mass spectrometry was applied to simultaneously determine the level of florfenicol and florfenicol amine in agricultural soils. Excellent linearity was achieved for florfenicol and florfenicol amine over a range of concentrations from 0.1-500 ?g/L with coefficients more than 0.99. Average recoveries at four different levels (0.005, 0.05, 0.5, and 5.0 mg/kg) for florfenicol and florfenicol amine ranged from 73.6-94.9% with relative standard deviations of 2.9-12.5%. The limits of detection for florfenicol and florfenicol amine in agricultural soils were 2.0 ?g/kg, and the limits of quantification were 6.0 ?g/kg. Based on this method, the degradation behavior of florfenicol and its metabolite florfenicol amine in three soils (Nanchang, Hangzhou, and Changchun) under sterilized and native conditions was investigated and the transformation rate of florfenicol amine from florfenicol was evaluated. PMID:25395188

Xu, Mingfei; Qian, Mingrong; Zhang, Hu; Ma, Junwei; Wang, Jianmei; Wu, Huizhen

2015-01-01

362

Mitochondrial DNA Variation and Stock Structure of Walleyes from Eastern Lake Huron: An Analysis of Contemporary and Historical Samples  

Microsoft Academic Search

The spatial and temporal distributions of mitochondrial DNA (mtDNA) haplotypes in walleyes Stizostedion vitreum captured from spawning stocks were examined to assess genetic population structure in eastern Lake Huron, Ontario. We also assessed mtDNA variation in pond-reared fish relative to that in their parental sources to determine whether cultured fish have the potential to influence mtDNA structure within and among

Michael H. Gatt; Dylan J. Fraser; Arunas P. Liskauskas; Moira M. Ferguson

2002-01-01

363

Evaluation of DNAstable for DNA storage at ambient temperature.  

PubMed

Preserving DNA is important for validation of prospective and retrospective analyses, requiring many expensive types of equipment (e.g., freezers and back-up generators) and energy. While freezing is the most common method for storing extracted DNA evidence or well-characterized DNA samples for validation studies, DNAstable (Biomatrica), a commercially available medium for room temperature storage of DNA extracts was evaluated in this study. Two groups of samples consisting of different DNA quantities were investigated, one ranging from 20 to 400 ng (group 1) and the other one ranging from 1.4 to 20 ng (group 2). The DNA samples with and without DNAstable were stored at four different temperatures [?25 °C (room temperature), -20 °C, 37 °C or 50 °C]. DNA degradation over several months was monitored by SYBR Green-based qPCR assays and by PCR amplification of the core CODIS STR markers for group 1 and 2 DNA samples, respectively. For the time points tested in this study (up to 365 days), the findings indicate that the -20 °C controls and the DNAstable protected samples at room temperature provided similar DNA recoveries that were higher compared to the unprotected controls kept at RT, 37 °C or 50 °C. These results suggest that DNAstable can protect DNA samples with effectiveness similar to that of the traditional -20 °C freezing method. In addition, extrapolations from accelerated aging experiments conducted at high temperatures support that DNAstable is an effective technology for preserving purified DNA at room temperature with a larger protective impact on DNA samples of low quantity (<20 ng). PMID:24315605

Howlett, Susanne E; Castillo, Hilda S; Gioeni, Lora J; Robertson, James M; Donfack, Joseph

2014-01-01

364

Polymorphism in mtDNA control region of Mizo-Mongloid Breast Cancer samples as revealed by PCR-RFLP analysis.  

PubMed

Abstract Mutations in mitochondrial D-loop region of DNA (mtDNA) may serve as a potential sensor for cellular DNA damage and marker for cancer development. We investigated the restriction fragment length polymorphism (RFLP) pattern of the D-loop region in the blood samples of breast cancer patients among Mizoram population. Significant differences were observed among breast cancer and healthy blood samples in the RFLP pattern using AluI, HaeIII and RsaI enzymes. Polymorphic information content (PIC - 0.258), band informativeness (?Ib - 3.283) and marker index (MI - 0.006) were highest in the case of RsaI enzyme. Our data suggest that the RsaI polymorphic site in the mitochondrial control region is an informative marker for breast cancer development in Mizo population. PMID:25431825

Ghatak, Souvik; Lallawmzuali, Doris; Mukherjee, Subhajit; Mawia, Lal; Pautu, Jeremy L; Kumar, Nachimuthu Senthil

2014-11-28

365

Optimization of STR locus enrichment for STR profiling of fragmented DNA.  

PubMed

DNA degradation is a major obstacle in gaining an accurate profile with standard DNA typing technology. Although alternative genotyping strategies such as mini-STRs and SNPs have proven to be more successful in profiling degraded DNA, these approaches also have limitations. Here, we show that locus enrichment by hybridization of degraded genomic DNA with an STR locus-specific biotinylated oligonucleotide is a powerful approach to overcome problems in STR typing of highly degraded DNA. An experimental investigation of factors affecting the efficiency of this method indicates that the choice of primer and molar ratio of primers to genomic DNA are critical factors in improving enrichment of the STR locus before genotyping with multiplex kits. In addition, we find that indirect capture rather than direct capture with magnetic beads yields better enrichment efficiency for STR locus enrichments. Using these strategies, we demonstrate an improvement in STR typing of DNA from cultured cells damaged by exposure to sunlight or UV. We suggest that this approach could be applied to highly degraded forensic samples alone or in combination with mini-STRs. PMID:25142119

Ham, Seon-Kyu; Kim, Se-Yong; Ahn, Jang-Won; Seo, Bo Young; Woo, Kwang-Man; Choi, Cheol Yong; Lee, Seung-Hwan

2014-11-01

366

Thermal degradation chemistry of poly[bis(phenoxy)phosphazene  

E-print Network

the model compound studies and from the degradation of PBPP suggests that phenyl migration occurs at some stage during degradation. Some of these findings are in disagreement with previously reported studies. P solution-state and solid-state NMR... CONCLUSIONS REFERENCES Y ITA Dynamic degradation of PBPP samples ~othermal degradation of model compound samples. P Solution-State NMR characterization of PBPP samples. Isothermal degradation of PBPP samples Solution-State hydrolysis of PBPP samples...

Maynard, Shawn Joseph

1989-01-01

367

Effects of DNA Extraction Procedures on Bacteroides Profiles in Fecal Samples From Various Animals Determined by Terminal Restriction Fragment Length Polymorphism Analysis  

EPA Science Inventory

A major assumption in microbial source tracking is that some fecal bacteria are specific to a host animal, and thus provide unique microbial fingerprints that can be used to differentiate hosts. However, the DNA information obtained from a particular sample may be biased dependi...

368

Nondestructive sampling of insect DNA from defensive H. M. DONALD,* C. W. WOOD,* K. M. BENOWITZ,* R. A. JOHNSON, E. D. BRODIE III* and  

E-print Network

Nondestructive sampling of insect DNA from defensive secretion H. M. DONALD,* C. W. WOOD,* K. M and Wildlife Resources, University of Idaho, Moscow, ID 83844, USA Abstract Nondestructive techniques to obtain a nondestructive technique. A secretion containing haemolymph was obtained from Bolitotherus cornutus (the forked

Brodie III, Edmund D.

369

Degradation of endogenous and exogenous genes of roundup-ready soybean during food processing.  

PubMed

Roundup-Ready soybeans have been genetically modified to resist the effects of the herbicidal glyphosate and have become the most prevalent transgenic crop in the world. In this work, Roundup-Ready soybeans were used as raw material to study the effects of critical processing procedures such as grinding, cooking, blending, homogenization, sterilization, and spray-drying on the length of DNA fragments of an endogenous gene (lectin) and an exogenous gene (epsps) examined in material from three soybean foods of bean curd, soy milk, and soy powder and from samples taken during their processing. The results showed that various processing procedures caused degradations of both the endogenous and exogenous genes to different degrees. In the grinding procedure, endogenous gene DNA was degraded from 1883 to approximately 836 bp, and exogenous gene DNA was degraded from 1512 to approximately 408 bp. In the blending and squeeze-molding procedures, exogenous gene DNA was also degraded from about 408 to 190 bp, but there was no obvious action on the endogenous gene. After the endogenous and exogenous genes had been degraded to some degree, such as 836 and 408 bp, respectively, they were not evidently affected by cooking procedure at 100 degrees C for 15 min. However, the endogenous gene was further considerably degraded from around 836 to 162 bp in the sterilization procedure at 121 degrees C for 30 s. The effect of the homogenization step on endogenous and exogenous genes was similar to that of the cooking procedure. The coagulation procedure, principally a biochemical reaction, did not greatly affect the exogenous gene but did affect endogenous gene, reducing DNA size from about 836 to 407 bp. Furthermore, the spray-drying procedure, a process of physical shearing, high temperature, and sudden high pressure, distinctly caused degradation of both the lectin and epsps genes, rapidly decreasing the sizes from about 836 to 162 bp for the endogenous gene and from about 408 to 190 bp for the exogenous gene. PMID:16366721

Chen, Ying; Wang, Yuan; Ge, Yiqiang; Xu, Baoliang

2005-12-28

370

Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA.  

PubMed

Analyses of degraded DNA are typically hampered by contamination, especially when employing universal primers such as commonly used in environmental DNA studies. In addition to false-positive results, the amplification of contaminant DNA may cause false-negative results because of competition, or bias, during the PCR. In this study, we test the utility of human-specific blocking primers in mammal diversity analyses of ancient permafrost samples from Siberia. Using quantitative PCR (qPCR) on human and mammoth DNA, we first optimized the design and concentration of blocking primer in the PCR. Subsequently, 454 pyrosequencing of ancient permafrost samples amplified with and without the addition of blocking primer revealed that DNA sequences from a diversity of mammalian representatives of the Beringian megafauna were retrieved only when the blocking primer was added to the PCR. Notably, we observe the first retrieval of woolly rhinoceros (Coelodonta antiquitatis) DNA from ancient permafrost cores. In contrast, reactions without blocking primer resulted in complete dominance by human DNA sequences. These results demonstrate that in ancient environmental analyses, the PCR can be biased towards the amplification of contaminant sequences to such an extent that retrieval of the endogenous DNA is severely restricted. The application of blocking primers is a promising tool to avoid this bias and can greatly enhance the quantity and the diversity of the endogenous DNA sequences that are amplified. PMID:21988749

Boessenkool, Sanne; Epp, Laura S; Haile, James; Bellemain, Eva; Edwards, Mary; Coissac, Eric; Willerslev, Eske; Brochmann, Christian

2012-04-01

371

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

PubMed Central

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

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

2014-01-01

372

SOIL DEGRADATION  

Technology Transfer Automated Retrieval System (TEKTRAN)

Soil degradation can be defined as loss in the quality or productivity of soil, and is often the result of human activities, such as agriculture, deforestation, mining, waste disposal, or chemical spills. Degradation is attributed to changes in soil nutrient status, biota, loss of organic matter, d...

373

A Study on the Effects of Degradation and Template Concentration on the Amplification Efficiency of the STR Miniplex Primer Sets  

Microsoft Academic Search

ABSTRACT: In forensic DNA analysis, the samples recovered from the crime scene are often highly degraded leading to poor PCR amplification of the larger sized STR loci. To avoid this problem, we have developed STR markers with redesigned primer sequences called “Miniplexes” to produce smaller amplicons. To assess the effectiveness of these kits, we have tested these primer sets with

Denise T. Chung; Ji?í Drábek; Kerry L. Opel; John M. Butler; Bruce R. McCord

2004-01-01

374

DNA in ancient bone - where is it located and how should we extract it?  

PubMed

Despite the widespread use of bones in ancient DNA (aDNA) studies, relatively little concrete information exists in regard to how the DNA in mineralised collagen degrades, or where it survives in the material's architecture. While, at the macrostructural level, physical exclusion of microbes and other external contaminants may be an important feature, and, at the ultrastructural level, the adsorption of DNA to hydroxyapatite and/or binding of DNA to Type I collagen may stabilise the DNA, the relative contribution of each, and what other factors may be relevant, are unclear. There is considerable variation in the quality of DNA retrieved from bones and teeth. This is in part due to various environmental factors such as temperature, proximity to free water or oxygen, pH, salt content, and exposure to radiation, all of which increase the rate of DNA decay. For example, bone specimens from sites at high latitudes usually yield better quality DNA than samples from temperate regions, which in turn yield better results than samples from tropical regions. However, this is not always the case, and rates of success of DNA recovery from apparently similar sites are often strikingly different. The question arises as to whether this may be due to post-collection preservation or just an artefact of the extraction methods used in these different studies? In an attempt to resolve these questions, we examine the efficacy of DNA extraction methods, and the quality and quantity of DNA recovered from both artificially degraded, and genuinely ancient, but well preserved, bones. In doing so we offer hypotheses relevant to the DNA degradation process itself, and to where and how the DNA is actually preserved in ancient bone. PMID:21855309

Campos, Paula F; Craig, Oliver E; Turner-Walker, Gordon; Peacock, Elizabeth; Willerslev, Eske; Gilbert, M Thomas P

2012-01-20

375

Development of a new real-time polymerase chain reaction assay to detect Duck adenovirus A DNA and application to samples from Swiss poultry flocks.  

PubMed

Between 2008 and 2012, commercial Swiss layer and layer breeder flocks experiencing problems in laying performance were sampled and tested for infection with Duck adenovirus A (DAdV-A; previously known as Egg drop syndrome 1976 virus). Organ samples from birds sent for necropsy as well as blood samples from living animals originating from the same flocks were analyzed. To detect virus-specific DNA, a newly developed quantitative real-time polymerase chain reaction method was applied, and the presence of antibodies against DAdV-A was tested using a commercially available enzyme-linked immunosorbent assay. In 5 out of 7 investigated flocks, viral DNA was detected in tissues. In addition, antibodies against DAdV-A were detected in all of the flocks. PMID:24590667

Schybli, Martina; Sigrist, Brigitte; Hess, Michael; van Leerdam, Bart; Hoop, Richard K; Vögtlin, Andrea

2014-03-01

376

Effect of antibodies to double stranded DNA, purified from serum samples of patients with active systemic lupus erythematosus, on the glomerular mesangial cells.  

PubMed Central

Polyclonal antibodies to double stranded DNA (dsDNA) purified from pooled serum samples of patients with systemic lupus erythematosus (SLE) exerted cytotoxic effects on cultured rat mesangial cells. At concentrations from 5 to 150 IU/ml, antibodies to dsDNA inhibited the incorporation of thymidine labelled with 3H into rat mesangial cells in a dose response manner after three days of culture. In contrast, normal human IgG (1 mg/ml), heat aggregated human IgG (1 mg/ml), N-formyl-methionyl-leucyl-phenylalanine (1 x 10(-7) mol/l), tumour necrosis factor alpha (16 U/ml), lipopolysaccharides (1 microgram/ml), 4 beta-phorbol-12 beta-myristate-13 alpha-acetate (PMA) (20 ng/ml), interleukin 1 beta (10 U/ml), and 20% v/v phytohaemagglutinin stimulated mononuclear cell supernatant showed no significant effect on these cells. Anticardiolipin antibody, another autoantibody purified from the serum of patients with SLE, also inhibited the proliferation of rat mesangial cells but to a lesser extent. In the presence of antibodies to dsDNA (100 IU/ml), the mesangial cells became spherical and clustered together, which was very different from the original stellate appearance. These autoantibodies also depolarised the membrane potential of mesangial cells. Antibodies to dsDNA decreased the syntheses of prostaglandin E2, 6-keto-prostaglandin F1 alpha and thromboxane B2 by mesangial cells. In an in vivo study, the antibodies to dsDNA showed a strong affinity for the glomeruli when intravenously injected into rats. These results suggest that the nephrotropic antibodies to dsDNA can directly damage the glomerular mesangial cells in addition to the formation of immune complexes with DNA which may cause kidney inflammation and tissue destruction. Images PMID:1550397

Tsai, C Y; Wu, T H; Sun, K H; Yu, C L

1992-01-01

377

ISOLATION OF EXTREMELY AT-RICH GENOMIC DNA AND ANALYSIS OF GENES ENCODING CARBOHYDRATE-DEGRADING ENZYMES FROM ORPINOMYCES SP. STRAIN PC-2  

Technology Transfer Automated Retrieval System (TEKTRAN)

An effective method for extraction of intact genomic DNA from extremely AT-rich anaerobic fungus Orpinomyces sp. strain PC-2 has been developed. This procedure involves removal of glycogenlike storage polysaccharides using repeated hexadecyltrimethylammonium bromide (CTAB) and high salt wash. The ...

378

DNA typing for the identification of old skeletal remains from Korean War victims.  

PubMed

The identification of missing casualties of the Korean War (1950-1953) has been performed using mitochondrial DNA (mtDNA) profiles, but recent advances in DNA extraction techniques and approaches using smaller amplicons have significantly increased the possibility of obtaining DNA profiles from highly degraded skeletal remains. Therefore, 21 skeletal remains of Korean War victims and 24 samples from biological relatives of the supposed victims were selected based on circumstantial evidence and/or mtDNA-matching results and were analyzed to confirm the alleged relationship. Cumulative likelihood ratios were obtained from autosomal short tandem repeat, Y-chromosomal STR, and mtDNA-genotyping results, and mainly confirmed the alleged relationship with values over 10?. The present analysis emphasizes the value of mini- and Y-STR systems as well as an efficient DNA extraction method in DNA testing for the identification of old skeletal remains. PMID:20456584

Lee, Hwan Young; Kim, Na Young; Park, Myung Jin; Sim, Jeong Eun; Yang, Woo Ick; Shin, Kyoung-Jin

2010-11-01

379

DNA barcoding for species identification from dried and powdered plant parts: A case study with authentication of the raw drug market samples of Sida cordifolia.  

PubMed

The majority of the plant materials used in herbal medicine is procured from the markets in the form of dried or powdered plant parts. It is essential to use authentic plant materials to derive the benefits of herbal medicine. However, establishing the identity of these plant materials by conventional taxonomy is extremely difficult. Here we report a case study in which the species identification of the market samples of Sida cordifolia was done by DNA barcoding. As a prelude to species identification by DNA barcoding, 13 species of Sida were collected, and a reference DNA barcode library was developed using rbcL, matK, psbA-trnH and ITS2 markers. Based on the intra-species and inter-species divergence observed, psbA-trnH and ITS2 were found to be the best two-marker combination for species identification of the market samples. The study showed that none of the market samples belonged to the authentic species, S. cordifolia. Seventy-six per cent of the market samples belonged to other species of Sida. The predominant one was Sida acuta (36%) followed by S. spinosa (20%), S. alnifolia (12%), S. scabrida (4%) and S. ravii (4%). Such substitutions may not only fail to give the expected therapeutic effect, but may also give undesirable effects as in case of S. acuta which contains a 6-fold higher amount of ephedrine compared to the roots of S. cordifolia. The remaining 24% of the samples were from other genera such as Abutilon sp. (8%), Ixonanthes sp., Terminalia sp., Fagonia sp., and Tephrosia sp. (4% each). This observation is in contrast to the belief that medicinal plants are generally substituted or adulterated with closely related species. The current study strongly suggests that the raw drug market samples of herbal medicines need to be properly authenticated before use, and DNA barcoding has been found to be suitable for this purpose. PMID:25596347

Vassou, Sophie Lorraine; Kusuma, G; Parani, Madasamy

2015-03-15

380

Original article Ribosomal DNA and chloroplast DNA  

E-print Network

Original article Ribosomal DNA and chloroplast DNA polymorphisms in a mixed stand of Quercus robur in western France. The ribosomal DNA repeat was characterized by a high level of length polymorphism; while chloro- plast DNA in our sample was nearly fixed at 2 previously identified polymorphic regions. Overall

Boyer, Edmond

381

Highly accurate SNP genotyping from historical and low-quality samples  

Microsoft Academic Search

Historical and other poor-quality samples are often necessary for population genetics, conservation, and forensics studies. Although there is a long history of using mtDNA from such samples, obtaining and genotyping nuclear loci have been considered difficult and error-prone at best, and impossible at worst. The primary issues are the amount of nuclear DNA available for genotyping, and the degradation of

PHILLIP A. MORIN; MELISSA MCCARTHY

2007-01-01

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DNA Barcoding  

NSDL National Science Digital Library

This is a two-part animation. Â?DNA Barcoding, Part 1,Â? provides an overview of how DNA barcoding of animals can be used to identify an unknown sample or discover a new species. Cytochrome c oxidase subunit 1 (COI) is found in the mitochondria as part of the electron transport chain. The COI gene is used for DNA barcoding. Just like a barcode on an item in a grocery store identifies a product, a DNA barcode (determined by DNA sequencing) is used to identify species. Part 1 run time: 1 minute, 40 seconds. Â?DNA Barcoding, Part 2Â? details how small tissue samples are used for DNA barcoding, including a review of the laboratory and bioinformatics steps used in barcoding: DNA purification, polymerase chain reaction (PCR), agarose gel electrophoresis, DNA sequencing and analysis, and DNA sequence identification using the Basic Local Alignment Search Tool (BLAST) or the Barcode of Life Database (BOLD). Part 2 run time: 4 minutes, 15 seconds. Animation is closed captioned.

2012-10-22