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1

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

Microsoft Academic Search

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

Bruce E Deagle; J Paige Eveson; Simon N Jarman

2006-01-01

2

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

3

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

PubMed Central

Ancient and archival DNA samples are valuable resources for the study of diverse historical processes. In particular, museum specimens provide access to biotas distant in time and space, and can provide insights into ecological and evolutionary changes over time. However, archival specimens are difficult to handle; they are often fragile and irreplaceable, and typically contain only short segments of denatured DNA. Here we present a set of tools for processing such samples for state-of-the-art genetic analysis. First, we report a protocol for minimally destructive DNA extraction of insect museum specimens, which produced sequenceable DNA from all of the samples assayed. The 11 specimens analyzed had fragmented DNA, rarely exceeding 100 bp in length, and could not be amplified by conventional PCR targeting the mitochondrial cytochrome oxidase I gene. Our approach made these samples amenable to analysis with commonly used next-generation sequencing-based molecular analytic tools, including RAD-tagging and shotgun genome re-sequencing. First, we used museum ant specimens from three species, each with its own reference genome, for RAD-tag mapping. Were able to use the degraded DNA sequences, which were sequenced in full, to identify duplicate reads and filter them prior to base calling. Second, we re-sequenced six Hawaiian Drosophila species, with millions of years of divergence, but with only a single available reference genome. Despite a shallow coverage of 0.37±0.42 per base, we could recover a sufficient number of overlapping SNPs to fully resolve the species tree, which was consistent with earlier karyotypic studies, and previous molecular studies, at least in the regions of the tree that these studies could resolve. Although developed for use with degraded DNA, all of these techniques are readily applicable to more recent tissue, and are suitable for liquid handling automation. PMID:24828244

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

2014-01-01

4

Early Necrotic DNA Degradation  

PubMed Central

The structure of DNA breaks in early necrosis was analyzed and compared with apoptotic DNA degradation using in vivo and cell culture models. Early necrosis (1 hour after cell death) was produced in vivo by the freezing-thawing of rat thymus and in cell culture of Jurkat cells. Apoptosis was induced in the same cell types using dexamethasone for thymus and staurosporine for Jurkat cells. Selective detection of double-strand DNA breaks with blunt ends was performed by in situ ligation. Blunt-ended breaks bearing 5? phosphates were detected in apoptotic but not in early necrotic cells. Pretreatment of apoptotic and necrotic tissue with Klenow enzyme with or without added dNTPs reduced all 3? or 5? overhangs to blunt ends. Subsequent in situ ligation with blunt-ended probes revealed no 3? overhangs in necrotic cells. However double-strand cuts with 5? overhangs were abundant in necrotic DNA. 5? Overhangs were also detected in apoptotic cells. Presence of exclusively 5? overhangs in early necrosis with absence of a variety of possible DNA ends, suggests the existence of a specific orderly mechanism of DNA degradation. PMID:12707041

Didenko, Vladimir V.; Ngo, Hop; Baskin, David S.

2003-01-01

5

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

PubMed

There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition. However, a system which can also assess the extent of degradation in a forensic sample will be a useful tool for DNA analysts. Possessing this information prior to genotyping will allow an analyst to more informatively make downstream decisions for the successful typing of a forensic sample without unnecessarily consuming DNA extract. Real-time PCR provides a reliable method for determining the amount and quality of amplifiable DNA in a biological sample. Alu are Short Interspersed Elements (SINE), approximately 300bp insertions which are distributed throughout the human genome in large copy number. The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human genome, which minimizes the individual specific variation possible when using a multi-copy target quantification system. This study utilizes two independent retrotransposon genomic targets to obtain quantification of an 80bp "short" DNA fragment and a 207bp "long" DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™. The ratio of the two quantitation values provides a "Degradation Index", or a qualitative measure of a sample's extent of degradation. The Degradation Index was found to be predictive of the observed loss of STR markers and alleles as degradation increases. Use of a synthetic target as an internal positive control (IPC) provides an additional assessment for the presence of PCR inhibitors in the test sample. In conclusion, a DNA based qualitative/quantitative/inhibition assessment system that accurately predicts the status of a biological sample, will be a valuable tool for deciding which DNA test kit to utilize and how much target DNA to use, when processing compromised forensic samples for DNA testing. PMID:25212510

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

2014-11-01

6

Evaluation of degradation in DNA from males with a quantitative gender typing, endpoint PCR multiplex.  

PubMed

Evidentiary samples submitted to a forensic DNA laboratory occasionally yield DNA that is degraded. Samples of intact chromosomal DNA (both nuclear and mitochondrial) were subjected to a heating protocol to induce DNA degradation. The DNAs were then analyzed using a multiplex PCR assay that amplifies targets of low and high molecular weight on the X/Y and mitochondrial chromosomes. If degradation is random, the amplification of larger DNA targets should be more adversely affected by degradation than smaller targets. In nuclear and mitochondrial DNA from a male donor, exhibiting degradation, DNA quantity estimates based upon higher molecular weight amplicons (HMW) are significantly lower than estimates made using low molecular weight (LMW) Q-TAT amplicons. DNA degradation estimated using this approach correlated well with actual fluorescence associated with HMW and LMW STR alleles amplified from the same genomic DNA templates. Q-TAT is thus useful not only as a quantitation tool, but also as an indicator of template degradation. PMID:25537731

Smith, Byron C; Vandegrift, Emily; Fuller, Valerie Mattimore; Allen, Robert W

2015-03-01

7

Comparison of fluorometric and spectrophotometric DNA quantification for real-time quantitative PCR of degraded DNA  

Microsoft Academic Search

Isogenic NK603 DNA was degraded by sonication or heat and quantified using A260 and two fluorescent dye methods. Quantitative PCR (qPCR) experiments were conducted by amplifying an SSIIb-3 endogenous control and an NK603 transgene in untreated, sonicated, and heat treated samples. qPCR reactions on sonicated DNA samples, based on A260 quantification, provided 0.125%, 1.14% and 2.15% NK603; while heat treated

Luke A. Shokere; Marcia J. Holden; G. Ronald Jenkins

2009-01-01

8

Radiation-induced degradation of DNA bases  

NASA Astrophysics Data System (ADS)

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

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

1999-01-01

9

DNA degradation of genetically modified cottonseed meal during feed processing.  

PubMed

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

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

2013-01-01

10

Quantifiler(®) Trio Kit and forensic samples management: A matter of degradation.  

PubMed

DNA collected from crime scenes may have experienced different levels of degradation. This is mainly due to sample exposure to different environmental factors. The impact of DNA degradation on short tandem repeat (STR) profiling can lead to partial or null information and in some cases, the identification of the trace may fail. The availability of a system enabling the assessment not only of the quantity of the DNA but also of its quality in terms of degradation would result in shorter time for sample processing, more reliable identifications and cost reduction by predicting the quality of the DNA profiles prior to STR analysis. We report here a study on 181 selected degraded DNA samples extracted from real crime scene evidence. The selected samples were processed by combining the use of a new commercial quantification kit (Quantifiler(®) Trio) with a new 24 marker multiplex PCR amplification kit (Globalfiler(®) Kit). Applying different statistical analyses we investigated the reliability of the Degradation Index provided by the Quantifiler(®) Trio in determining the level of DNA degradation in a forensic sample. This useful information can be used to predict the quality of the profile obtained after STR amplification. The combination of such a quantification kit with different PCR protocols allowed us to define practical guidelines for processing degraded forensic DNA samples with a simplified and comprehensive approach. PMID:25544252

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

2015-05-01

11

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

PubMed Central

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

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

2009-01-01

12

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...

13

[Application of repair enzymes to improve the quality of degraded DNA templates for PCR amplification].  

PubMed

PCR amplification of severely degraded DNA, including ancient DNA, forensic samples, and preparations from deeply processed foodstuffs, is a serious problem. Living organisms have a set of enzymes to repair lesions in their DNA. In this work, we have developed and characterized model systems of degraded high-molecular-weight DNA with a predominance of different types of damage. It was shown that depurination and oxidation of the model plasmid DNA template led to a decrease in the PCR efficiency. A set of enzymes performing a full cycle of excision repair of some lesions was determined. The treatment of model-damaged substrates with this set of enzymes resulted in an increased PCR product yield as compared with that of the unrepaired samples. PMID:25757334

Dovgerd, A P; Zharkov, D O

2014-01-01

14

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

15

Analysis of 9 mitochondrial SNP's from samples with trace Amount of DNA  

Microsoft Academic Search

Forensic scientists often use nuclear STR loci for identification. But sometimes it is difficult to get successful results from nuclear DNA, especially when the DNA in a sample is degraded or the amount is not sufficient. In these cases forensic scientists use mitochondrial DNA (mtDNA). The aim of this work is to genotype of the 9 mitochondrial SNP sites (3010,

A. M. Ölçen; G. Filo?lu; H. Altunçul; S. Erdem; Ö. Bülbül

16

Sex identification of ancient DNA samples using a microfluidic device.  

PubMed

Ancient DNA is the name given to the degraded, fragmented, and chemically damaged biomolecules that can be recovered from archaeological remains of plants, animals, and humans. Where ancient human DNA has survived at archaeological sites, it can give valuable information and is especially useful for its potential to identify kinship, population affinities, pathogens, and biological sex. Here, we describe the operation of a microfluidic device for the sex identification of ancient DNA samples using an efficient sample handling process. DNA is extracted from powdered bone samples and abasic sites labeled with biotin. Streptavidin-coated superparamagnetic particles are used to isolate the labeled DNA prior to amplification of the Amelogenin sex marker. PMID:25673485

Shaw, Kirsty J; Brown, Keri A; Brown, Terence A; Haswell, Stephen J

2015-01-01

17

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...

18

Methods and reagents. Degraded DNA and gel tornados.  

PubMed

Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses a case of inexplicable DNA degradation and tornados seen in agarose gels. For details on how to partake in the newsgroup, see the accompanying box. PMID:9149534

Hengen, P N

1997-04-01

19

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

20

Filter Paper for Preservation, Storage, and Distribution of Insect and Pathogen DNA Samples  

Microsoft Academic Search

Proper DNA storage is critical for studies involving genetic analysis of insects and for molecular diagnostics of pathogens carried by them. Molecular surveillance of pathogens carried by insects can involve screening of thousands of insect DNA samples. Problems with storage and degradation of these samples can arise. In this study, a simple Þlter paper-based method for storage and preservation of

Carrie B. Owens; Allen L. Szalanski

2005-01-01

21

Amplification of bleomycin-mediated degradation of DNA.  

PubMed

Three simple and independent tests have been introduced for studying the effect of DNA intercalating compounds on the bleomycin-mediated digestion of DNA in vitro. These methods are based on hyperchromic changes of DNA solution, changes in viscosity of DNA solution, and HPLC quantitative analysis of the four bases released from digested DNA. All three tests give comparable results. However, the viscometric method is technically the simplest and at the same time the most sensitive. The amplification of the bleomycin-mediated degradation of DNA by three unfused heteropolyaromatic intercalator molecules, namely N-[2''-(dimethylamino)ethyl]-4-thien-2'-ylpyrimidin-2-amine (1N), N,N-dimethyl-2-[(4'-thien-2''-ylpyrimidin-2'-yl)thio] ethylamine (1S), and newly synthesized 2,5-bis[2'-[[2''-(dimethylamino)ethyl]thio]pyrimidin-4'yl]thiophene (2) correlates well with the respective DNA binding constants for these compounds and is concentration dependent. The amplification activity of these compounds increases with increasing concentrations. The strongly binding compound 2 is the best amplifier of bleomycin in vitro found so far. Fused heteropolyaromatic systems, like ethidium bromide, are modest amplifiers of bleomycin at low concentrations but strongly inhibit the bleomycin chemistry at high concentrations. PMID:2441055

Strekowski, L; Strekowska, A; Watson, R A; Tanious, F A; Nguyen, L T; Wilson, W D

1987-08-01

22

Geminin, an Inhibitor of DNA Replication, Is Degraded during Mitosis  

Microsoft Academic Search

We describe a novel 25 kDa protein, geminin, which inhibits DNA replication and is degraded during the mitotic phase of the cell cycle. Geminin has a destruction box sequence and is ubiquitinated anaphase-promoting complex (APC) in vitro. In synchronized HeLa cells, geminin is absent during G1 phase, accumulates during S, G2, and M phases, and disappears at the time of

Thomas J McGarry; Marc W Kirschner

1998-01-01

23

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

24

DNA compaction induced by a cationic polymer or surfactant impact gene expression and DNA degradation.  

PubMed

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

25

Field scale evaluation of bovine-specific DNA as an indicator of tissue degradation during cattle mortality composting.  

PubMed

Currently, mortality compost is managed by temperature as extent of tissue degradation is difficult to assess. In the present study, field-scale mortality compost was constructed with composted brain tissue (Brain) and compost adjacent to brain tissue (CAB) sampled over 230 d. Following genomic DNA extraction, bovine-specific mitochondrial DNA (Mt-DNA) and bacterial 16S rDNA fragments were quantified using real-time PCR. Genomic DNA yield of Brain and CAB decreased rapidly (89-98%) and stabilized after 7 d. Compared to d 0, Brain Mt-DNA rapidly decreased (84-91% reduction on d 7). In CAB, Mt-DNA dramatically increased until d 28 (up to 34,500 times) thereafter decreasing by 77-93% on d 112. Quantification of bovine Mt-DNA indicates tissue degradation was initially characterized by rapid decomposition and release of cell contents into surrounding compost matrix followed by further degradation of Mt-DNA by flourishing microorganisms. Consequently, bovine Mt-DNA copies in compost matrix were reliable indicators of tissue degradation. PMID:21316954

Xu, Weiping; Reuter, Tim; Xu, Yongping; Hsu, Yu-Hung; Stanford, Kim; McAllister, Tim A

2011-04-01

26

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

27

Physiochemical degradation of thermally aged hypalon glove samples  

Microsoft Academic Search

Attenuated total reflection (ATR) infrared spectra have been analyzed using multivariate curve resolution (MCR) to capture the chemistry of the thermal degradation in the aging of chlorosulfonated polyethylene (Hypalon®) glove samples. The analysis demonstrates the primary degradation pathways to be oxidation (formation of ketones and carboxylic acids), dehydrochlorination with formation of –C?C– groups, and polymer crosslinking with changes in the

Kennard V. Wilson; Bettina L. Smith; John M. Macdonald; Jon R. Schoonover; Julio M. Castro; Mark E. Smith; Michael E. Cournoyer; Rob Marx; Warren P. Steckle

2004-01-01

28

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

PubMed Central

In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the 13C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-13C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from 13C-enriched DNA and were designated “anthracene group 1.” Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP. PMID:21398486

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

2011-01-01

29

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

30

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

PubMed

In this paper we report on the identification of a section of mitochondrial DNA that can be used to identify the species of protected and illegally traded pythons of the genus Morelia. Successful enforcement of wildlife laws requires forensic tests that can identify the species nominated in the relevant legislation. The potentially degraded state of evidentiary samples requires that forensic investigation using molecular genetic species identification is optimized to interrogate small fragments of DNA. DNA was isolated from 35 samples of Morelia spilota from which the complete cytochrome b was sequenced. The ND6 gene was also sequenced in 32 of these samples. Additional DNA sequences were generated from 9 additional species of Morelia. The sequences were aligned by Geneious and imported into MEGA to create phylogenetic trees based on the entire complex of approximately 1,706 base pairs (bp). To mimic degraded DNA, which is usually found in forensic cases, short sub-sections of the full alignment were used to generate phylogenetic trees. The sub-sections that had the greatest DNA sequence information were in parts of the cytochrome b gene. Our results highlight that legislation is presently informed by inadequate taxonomy. We demonstrated that a 278 bp region of the cytochrome b gene recovered the topology of the phylogenetic tree found with the entire gene sequence and correctly identified species of Morelia with a high degree of confidence. The locus described in this report will assist in the successful prosecution of alleged illegal trade in python species. PMID:24915762

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

2014-09-01

31

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

32

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

33

Heat degradation of eukaryotic and bacterial DNA: an experimental model for paleomicrobiology  

PubMed Central

Background Theoretical models suggest that DNA degradation would sharply limit the PCR-based detection of both eukaryotic and prokaryotic DNA within ancient specimens. However, the relative extent of decay of eukaryote and prokaryote DNA over time is a matter of debate. In this study, the murine macrophage cell line J774, alone or infected with Mycobacterium smegmatis bacteria, were killed after exposure to 90°C dry heat for intervals ranging from 1 to 48 h in order to compare eukaryotic cells, extracellular bacteria and intracellular bacteria. The sizes of the resulting mycobacterial rpoB and murine rpb2 homologous gene fragments were then determined by real-time PCR and fluorescent probing. Findings The cycle threshold (Ct) values of PCR-amplified DNA fragments from J774 cells and the M. smegmatis negative controls (without heat exposure) varied from 26–33 for the J774 rpb2 gene fragments and from 24–29 for M. smegmatis rpoB fragments. After 90°C dry heat incubation for up to 48 h, the Ct values of test samples increased relative to those of the controls for each amplicon size. For each dry heat exposure time, the Ct values of the 146-149-bp fragments were lower than those of 746-747-bp fragments. During the 4- to 24-h dry heat incubation, the non-infected J774 cell DNA was degraded into 597-bp rpb2 fragments. After 48 h, however, only 450-bp rpb2 fragments of both non-infected and infected J774 cells could be amplified. In contrast, the 746-bp rpoB fragments of M. smegmatis DNA could be amplified after the 48-h dry heat exposure in all experiments. Infected and non-infected J774 cell DNA was degraded more rapidly than M. smegmatis DNA after dry heat exposure (ANOVA test, p?DNA was more resistant to dry-heat stress than eukaryotic DNA. Therefore, the detection of large, experimental, ancient mycobacterial DNA fragments is a suitable approach for paleomicrobiological studies. PMID:23009640

2012-01-01

34

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

PubMed

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

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

2014-01-01

35

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

36

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

37

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

38

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

39

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

40

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2014 CFR

...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

41

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

42

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

PubMed Central

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

2014-01-01

43

Modulation of eDNA Release and Degradation Affects Staphylococcus aureus Biofilm Maturation  

Microsoft Academic Search

Recent studies have demonstrated a role for Staphylococcus aureus cidA-mediated cell lysis and genomic DNA release in biofilm adherence. The current study extends these findings by examining both temporal and additional genetic factors involved in the control of genomic DNA release and degradation during biofilm maturation. Cell lysis and DNA release were found to be critical for biofilm attachment during

Ethan E. Mann; Kelly C. Rice; Blaise R. Boles; Jennifer L. Endres; Dev Ranjit; Lakshmi Chandramohan; Laura H. Tsang; Mark S. Smeltzer; Alexander R. Horswill; Kenneth W. Bayles; Adam J. Ratner

2009-01-01

44

Optimal storage conditions for highly dilute DNA samples: a role for trehalose as a preserving agent.  

PubMed

DNA extraction from trace samples or noninvasively collected samples often results in the recovery of low concentration solutions of DNA that are prone to DNA degradation or other loss. Because of the difficulty in obtaining such samples, and their potentially high value in wildlife and forensic studies, it is critical that optimal methods are employed for their long-term storage. We assessed the amplification yield of samples kept under different storage conditions with the addition of potential preserving agents. We stored dilutions of known concentration human placental DNA, and gorilla fecal DNA, under four conditions (+4 degrees C, -20 degrees C, -80 degrees C, dry at room temperature), and with three additives (Tris EDTA (TE) buffer, Hind III digested Lambda DNA, trehalose). The effectiveness of the treatment methods was tested at regular intervals using qPCR to assess the quantity of amplifiable DNA, and a PCR assay of a larger 757 bp fragment to evaluate the quality of that remaining DNA. The highest quantity of DNA remained in samples stored at -80 degrees C, regardless of storage additives, and those dried at room temperature in the presence of trehalose. Surprisingly, DNA quality was best preserved in the presence of trehalose, either dried or at -80 degrees C; significant quality loss occurred with -20 degrees C and +4 degrees C storage. PMID:16225214

Smith, Steve; Morin, Phillip A

2005-09-01

45

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

46

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

PubMed

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

Gryson, Nicolas

2010-03-01

47

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

48

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

49

Assessing a novel room temperature DNA storage medium for forensic biological samples.  

PubMed

The ability to properly collect, analyze and preserve biological stains is important to preserving the integrity of forensic evidence. Stabilization of intact biological evidence in cells and the DNA extracts from them is particularly important since testing is generally not performed immediately following collection. Furthermore, retesting of stored DNA samples may be needed in casework for replicate testing, confirmation of results, and to accommodate future testing with new technologies. A novel room temperature DNA storage medium, SampleMatrix™ (SM; Biomatrica, Inc., San Diego, CA), was evaluated for stabilizing and protecting samples. Human genomic DNA samples at varying amounts (0.0625-200 ng) were stored dry in SM for 1 day to 1 year under varying conditions that included a typical ambient laboratory environment and also through successive freeze-thaw cycles (3 cycles). In addition, spiking of 1-4 × SM into samples prior to analysis was performed to determine any inhibitory effects of SM. Quantification of recovered DNA following storage was determined by quantitative PCR or by agarose gel electrophoresis, and evaluation of quantitative peak height results from multiplex short tandem repeat (STR) analyses were performed to assess the efficacy of SM for preserving DNA. Results indicate no substantial differences between the quality of samples stored frozen in liquid and those samples maintained dry at ambient temperatures protected in SM. For long-term storage and the storage of low concentration samples, SM provided a significant advantage over freezer storage through higher DNA recovery. No detectable inhibition of amplification was observed at the recommended SM concentration and complete profiles were obtained from genomic DNA samples even in the presence of higher than recommended concentrations of the SM storage medium. The ability to stabilize and protect DNA from degradation at ambient temperatures for extended time periods could have tremendous impact in simplifying and improving sample storage conditions and requirements. The current work focuses on forensics analysis; however this technology is applicable to all endeavors requiring storage of DNA. PMID:21324769

Lee, Steven B; Clabaugh, Kimberly C; Silva, Brie; Odigie, Kingsley O; Coble, Michael D; Loreille, Odile; Scheible, Melissa; Fourney, Ron M; Stevens, Jesse; Carmody, George R; Parsons, Thomas J; Pozder, Arijana; Eisenberg, Arthur J; Budowle, Bruce; Ahmad, Taha; Miller, Russell W; Crouse, Cecelia A

2012-01-01

50

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

PubMed

STR genotyping from degraded DNA samples requires genetic profiles to be obtained from DNA fragments no bigger than 200-300 bp. It requires the use of miniSTRs, which are smaller than the STRs used in standard typing. This paper reviews recent advances in miniSTR genotyping, beginning with a brief introduction to the processes involved in DNA fragmentation and how it hinders standard STR genotyping before proceeding further to the loci included in the main DNA databases and finishing with the International Workgroups' recommended design strategies for developing miniSTR reactions. The results of the efforts of many laboratories achieving different STR multiplexes and patents are also described and compared. Finally, a consideration of the perspectives for the future in this area is presented. PMID:21619547

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

2011-08-01

51

A minimalist barcode can identify a specimen whose DNA is degraded  

Microsoft Academic Search

A DNA barcode based on 650 bp of mitochondrial gene cytochrome c oxidase I is proving to be highly functional in species identification for various animal groups. However, DNA degradation complicates the recovery of a full-length barcode from many museum speci- mens. Here we explore the use of shorter barcode sequences for identification of such spec- imens. We recovered short

MEHRDAD HAJIBABAEI; M. A LEX SMITH; DANIEL H. JANZEN; JOSEPHINE J. RODRIGUEZ; JAMES B. W HITFIELD; PAUL D. N. H EBERT

2006-01-01

52

Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair.  

PubMed

Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3' side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4(Cdt2). Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase ? and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4(Cdt2) for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase ? to the damage site and completion of gap-filling DNA synthesis during the final stage of NER. PMID:25483071

Han, Chunhua; Wani, Gulzar; Zhao, Ran; Qian, Jiang; Sharma, Nidhi; He, Jinshan; Zhu, Qianzheng; Wang, Qi-En; Wani, Altaf A

2015-04-01

53

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

54

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

PubMed

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

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

2013-07-01

55

Fish scales and SNP chips: SNP genotyping and allele frequency estimation in individual and pooled DNA from historical samples of Atlantic salmon (Salmo salar)  

PubMed Central

Background DNA extracted from historical samples is an important resource for understanding genetic consequences of anthropogenic influences and long-term environmental change. However, such samples generally yield DNA of a lower amount and quality, and the extent to which DNA degradation affects SNP genotyping success and allele frequency estimation is not well understood. We conducted high density SNP genotyping and allele frequency estimation in both individual DNA samples and pooled DNA samples extracted from dried Atlantic salmon (Salmo salar) scales stored at room temperature for up to 35 years, and assessed genotyping success, repeatability and accuracy of allele frequency estimation using a high density SNP genotyping array. Results In individual DNA samples, genotyping success and repeatability was very high (> 0.973 and?>?0.998, respectively) in samples stored for up to 35 years; both increased with the proportion of DNA of fragment size?>?1000 bp. In pooled DNA samples, allele frequency estimation was highly repeatable (Repeatability?=?0.986) and highly correlated with empirical allele frequency measures (Mean Adjusted R2?=?0.991); allele frequency could be accurately estimated in?>?95% of pooled DNA samples with a reference group of at least 30 individuals. SNPs located in polyploid regions of the genome were more sensitive to DNA degradation: older samples had lower genotyping success at these loci, and a larger reference panel of individuals was required to accurately estimate allele frequencies. Conclusions SNP genotyping was highly successful in degraded DNA samples, paving the way for the use of degraded samples in SNP genotyping projects. DNA pooling provides the potential for large scale population genetic studies with fewer assays, provided enough reference individuals are also genotyped and DNA quality is properly assessed beforehand. We provide recommendations for future studies intending to conduct high-throughput SNP genotyping and allele frequency estimation in historical samples. PMID:23819691

2013-01-01

56

Rapid cleanup of bacterial DNA from field samples  

Microsoft Academic Search

Polymerase chain reaction (PCR) is an in vitro enzymatic, synthetic method used to amplify specific DNA sequences from organisms. Detection of target DNA using gene probes allows for absolute identification not only of specific organisms, but also of genetic material in recombinant organisms. A major challenge facing detection of DNA from field samples is that they are almost sure to

Darrel E Menking; Peter A Emanuel; James J Valdes; Suzanne K Kracke

1999-01-01

57

Waikato DNA Sequencing Facility Please send samples to  

E-print Network

Waikato DNA Sequencing Facility Please send samples to: Attention: Waikato DNA Sequencing Facility quantitation are critical for successful sequencing reactions Ideally, the DNA supplied should be free-4757 Fax: (07) 838-4324 E-mail: sequence@waikato.ac.nz http://sequence.bio.waikato.ac.nz Name

Waikato, University of

58

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

59

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

PubMed Central

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis. PMID:12088997

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

2002-01-01

60

DNA degradation within mouse brain and dental pulp cells 72 hours postmortem?  

PubMed Central

In this study, we sought to elucidate the process of DNA degradation in brain and dental pulp cells of mice, within postmortem 0–72 hours, by using the single cell gel electrophoresis assay and professional comet image analysis and processing techniques. The frequency of comet-like cells, the percentage of tail DNA, tail length, tail moment, Olive moment and tail area increased in tandem with increasing postmortem interval. In contrast, the head radius, the percentage of head DNA and head area showed a decreasing trend. Linear regression analysis revealed a high correlation between these parameters and the postmortem interval. The findings suggest that the single cell gel electrophoresis assay is a quick and sensitive method to detect DNA degradation in brain and dental pulp cells, providing an objective and accurate new way to estimate postmortem interval.

Zheng, Jilong; Li, Xiaona; Shan, Di; Zhang, Han; Guan, Dawei

2012-01-01

61

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

PubMed

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

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

2014-06-13

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

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

EPA Science Inventory

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

64

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

Microsoft Academic Search

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present.

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

2011-01-01

65

Degradation of linear DNA by a strand-specific exonuclease activity in Xenopus laevis oocytes.  

PubMed Central

Linear DNA injected into Xenopus laevis oocyte nuclei recombines with high efficiency if homologous sequences are present at overlapping molecular ends. We found that injected linear DNA was degraded by a 5'----3' strand-specific exonuclease activity during incubation in the oocyte nucleus to leave a heterogeneous population of 3'-tailed molecules. Decreasing the concentration of DNA injected increased the heterogeneity and the average rate of degradation. The 3' tails created were relatively stable; among molecules persisting after overnight incubation, many had 3' tails intact to within 10 bases of the original ends. DNA molecules that were efficient substrates for homologous recombination in oocytes were also partially degraded, leaving 3' tails. We found no evidence for other potent nuclease activities. If molecules with recessed 3'-OH ends were injected, endogenous polymerase efficiently resynthesized complementary strands before degradation of the 5' tails occurred. 3'-tailed molecules are plausible intermediates in the initiation of homologous recombination events in Xenopus oocyte nuclei. Images PMID:2601699

Maryon, E; Carroll, D

1989-01-01

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

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

E-print Network

The influence of sample preparation and incubation sequence on in situ dry matter degradability was to determine the effect of sample preparation and incubation sequence on dry matter (DM) degradability of grass cannulated Friesian cows fed a silage, barley diet (80 : 20 ; DM basis) were used. Bags were incubated simul

Paris-Sud XI, Université de

68

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

PubMed Central

Polymeric gene-delivery vectors to achieve lack of toxicity and a balance between protection and DNA release remains a formidable challenge. Incorporating intracellular environment-responsive degradable bonds is an appreciable step toward developing safer transfection agents. In this study, novel, dual-degradable polycation copolymers (Pluronic-diacrylate [PA]–polyethyleneimine [PEI]–SS) were synthesized through the addition of low molecular weight (800 Da) PEI cross-linked with SS (PEI-SS) to PA. Three PA-PEI-SS copolymers (PA-PEI-SS1, 2, and 3) with different PEI-SS to Pluronic molar ratios were investigated and found to strongly condense plasmid DNA into positively charged nanoparticles with an average particle size of approximately 200 nm and to possess higher stability against DNase I digestion and sodium heparin. Disulfide and ester bonds of the copolymers were susceptible to intracellular redox conditions. In vitro experiments demonstrated that the PA-PEI-SS copolymers had significantly lower cytotoxicity and higher transfection efficiency in both BGC-823 and 293T cell lines than the controls of degradable PEI-SS and nondegradable 25 kDa PEI. Transfection activity was influenced by the PEI-SS content in the polymers and PA-PEI-SS1 showed the highest efficiency of the three copolymers. These studies suggest that these dual-degradable copolymers could be used as potential biocompatible gene delivery carriers. PMID:24109182

Zhang, Lifen; Chen, Zhenzhen; Li, Yanfeng

2013-01-01

69

PAH-degrading microbial consortium and its pyrene-degrading plasmids from mangrove sediment samples in Huian, China.  

PubMed

PAH-degrading microbial consortium and its pyrene-degrading plasmids were enriched from the sediment samples of Huian mangroves. The consortium YL showed degrading abilities of 92.1%, 87.6%, 92.3%, and 95.8% for pyrene, fluoranthene, phenanthrene, and fluoene at 50 mg l(-1) after 21 days incubation, respectively. The dynamics of pH changes in the cultures was consistent with that of PAH concentration change. Bacillus cereus Py5 and Bacillus megaterium Py6 were isolated from the consortium and observed consuming 65.8% and 33.7% of pyrene (50 mg l(-1)) within three weeks, respectively. The enriched Escherichia coli DH5alpha cells containing the plasmids of YL were demonstrated to degrade 85.7% of the original pyrene concentration at the 21st day. PMID:18501387

Lin, Yi; Cai, Li-Xi

2008-01-01

70

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

71

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

PubMed Central

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

Pollard, E C; Fugate, J K

1978-01-01

72

Targeted sampling of cementum for recovery of nuclear DNA from human teeth and the impact of common decontamination measures  

PubMed Central

Background Teeth are a valuable source of DNA for identification of fragmented and degraded human remains. While the value of dental pulp as a source of DNA is well established, the quantity and presentation of DNA in the hard dental tissues has not been extensively studied. Without this knowledge common decontamination, sampling and DNA extraction techniques may be suboptimal. Targeted sampling of specific dental tissues could maximise DNA profiling success, while minimising the need for laborious sampling protocols and DNA extraction techniques, thus improving workflows and efficiencies. We aimed to determine the location of cellular DNA in non-degraded human teeth to quantify the yield of nuclear DNA from cementum, the most accessible and easily sampled dental tissue, and to investigate the effect of a common decontamination method, treatment with sodium hypochlorite (bleach). We examined teeth histologically and subsequently quantified the yield of nuclear DNA from the cementum of 66 human third molar teeth. We also explored the effects of bleach (at varying concentrations and exposure times) on nuclear DNA within teeth, using histological and quantitative PCR methods. Results Histology confirmed the presence of nucleated cells within pulp and cementum, but not in dentine. Nuclear DNA yields from cementum varied substantially between individuals but all samples gave sufficient DNA (from as little as 20 mg of tissue) to produce full short tandem repeat (STR) profiles. Variation in yield between individuals was not influenced by chronological age or sex of the donor. Bleach treatment with solutions as dilute as 2.5% for as little as 1 min damaged the visible nuclear material and reduced DNA yields from cementum by an order of magnitude. Conclusions Cementum is a valuable, and easily accessible, source of nuclear DNA from teeth, and may be a preferred source where large numbers of individuals need to be sampled quickly (for example, mass disaster victim identification) without the need for specialist equipment or from diseased and degraded teeth, where pulp is absent. Indiscriminant sampling and decontamination protocols applied to the outer surface of teeth can destroy this DNA, reducing the likelihood of successful STR typing results. PMID:24139166

2013-01-01

73

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

74

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

75

Combined lipid\\/DNA extraction method for environmental samples  

Microsoft Academic Search

Previously, separate methods have been developed for the extraction and purification of lipids and DNA from soils and sediments. This paper describes a new method for the isolation of both lipids and DNA from the same environmental sample. This combined method is based on the Bligh and Dyer lipid extraction technique. Upon phase separation, lipids partition into the organic phase

S. R. Kehrmeyer; B. M. Applegate; H. C. Pinkart; D. B. Hedrick; D. C. White; G. S. Sayler

1996-01-01

76

Quantification of Cytosolic Plasmid DNA Degradation Using High-Throughput Sequencing: Implications for gene delivery  

PubMed Central

Background Although cytosolic DNA degradation plays an important role in decreasing transgene expression, the plasmid degradation pattern remains largely unexplored. Methods Illumina dye sequencing was employed to provide degradation site information for S1 and cytosolic nucleases. S1 nuclease provided a positive control for comparison between the agarose gel method and the sequencing approaches. Results The poly(A) region between the ?–lactamase gene and the CMV promoter was identified as most likely cut site for polyplex-treated cytosol. The second most likely site, at the 5’ end of the ?–lactamase gene, was identified by gel electrophoresis and sequencing. Additional sites were detected in the OriC region, the SV40/poly(A) region, the luciferase gene, and the CMV promoter. Sequence analysis of plasmid treated with cytosol from control cells showed the greatest cut activity in the OriC region, the ?–lactamase gene, and the poly(A) region following the luciferase gene. Additional regions of cut activity include the SV40 promoter and the ?–lactamase poly(A) termination sequence. Both cytosolic nucleases and the S1 nuclease showed substantial activity at the bacterial origin of replication (OriC). Conclusions High-throughput plasmid sequencing revealed regions of the luciferase plasmid DNA (pDNA) sequence that are sensitive to cytosolic nuclease degradation. This provides new targets for improving plasmid and/or polymer design to optimize the likelihood of protein expression. PMID:24700644

Rattan, Rahul; Bielinska, Anna U.; Banaszak, Mark M.

2014-01-01

77

Enhancement of oxidative DNA degradation by histidine: the role of stereochemical parameters.  

PubMed

The nicking of supercoiled DNA by H2O2 and ferrous iron has been studied in a variety of environmental conditions. The replicative form of phage fd DNA (fd RF DNA) was used for investigating the phenomenon. The rate of nicking was measured in 10 mM NaCl. The addition of 1 mM Tris-HCl buffer (pH 7.5) slowed down the rate of nicking, the addition of 0.1 mM histidine enhanced it. The simultaneous presence of 1 mM Tris-HCl buffer and of 0.1 mM histidine further enhanced the rate of nicking of fd RF DNA. Increasing the concentration of NaCl dramatically reduced the rate of the reaction. The degradation of fd RF DNA was determined as a function of the concentration of histidine (0-5 mM): the rate increases with concentration, reaches a maximum and then decreases. In the presence of histidine, increasing the concentration of Tris leads to a similar phenomenon. In the absence of histidine, Tris always quenches the degradation of DNA. Electron spin resonance measurements failed to detect an enhancement of the signal characteristic for the hydroxyl radical when histidine was added to the solution containing hydrogen peroxide and ferrous iron. When the nicking of DNA is achieved via the process of auto-oxidation of ferrous iron (i.e., in the absence of added H2O2), histidine only reduces the rate of reaction in a dose-dependent manner, in the explored range of concentrations. In the presence of H2O2 and ferrous iron, histidine enhances the rate of nicking of double-stranded DNA in its supercoiled as well as in its relaxed state, but fails to modify the rate of nicking of fd DNA when it is in its vegetative, single-stranded form. PMID:1379340

Marrot, L; Giacomoni, P U

1992-03-01

78

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

2015-03-01

79

Non-Destructive Sampling of Ancient Insect DNA  

PubMed Central

Background A major challenge for ancient DNA (aDNA) studies on insect remains is that sampling procedures involve at least partial destruction of the specimens. A recent extraction protocol reveals the possibility of obtaining DNA from past insect remains without causing visual morphological damage. We test the applicability of this protocol on historic museum beetle specimens dating back to AD 1820 and on ancient beetle chitin remains from permafrost (permanently frozen soil) dating back more than 47,000 years. Finally, we test the possibility of obtaining ancient insect DNA directly from non-frozen sediments deposited 3280-1800 years ago - an alternative approach that also does not involve destruction of valuable material. Methodology/Principal Findings The success of the methodological approaches are tested by PCR and sequencing of COI and 16S mitochondrial DNA (mtDNA) fragments of 77–204 base pairs (-bp) in size using species-specific and general insect primers. Conclusion/Significance The applied non-destructive DNA extraction method shows promising potential on insect museum specimens of historical age as far back as AD 1820, but less so on the ancient permafrost-preserved insect fossil remains tested, where DNA was obtained from samples up to ca. 26,000 years old. The non-frozen sediment DNA approach appears to have great potential for recording the former presence of insect taxa not normally preserved as macrofossils and opens new frontiers in research on ancient biodiversity. PMID:19337382

Thomsen, Philip Francis; Elias, Scott; Gilbert, M. Thomas P.; Haile, James; Munch, Kasper; Kuzmina, Svetlana; Froese, Duane G.; Holdaway, Richard N.; Willerslev, Eske

2009-01-01

80

Rapid extraction and preservation of genomic DNA from human samples  

PubMed Central

Simple and rapid extraction of human genomic DNA remains a bottle neck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab- and saliva samples. DNA is attracted on to a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle 4 microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for one month was demonstrated for captured DNA, facilitating straightforward collection, delivery and handling of genomic DNA in an environment-friendly protocol. PMID:23307121

Kalyanasundaram, D.; Kim, J.-H.; Yeo, W.-H.; Oh, K.; Lee, K.-H.; Kim, M.-H.; Ryew, S.-M.; Ahn, S.-G.; Gao, D.; Cangelosi, G. A.; Chung, J.-H.

2013-01-01

81

Preparation of DNA and nucleoprotein samples for AFM imaging  

PubMed Central

Sample preparation techniques allowing reliable and reproducible imaging of DNA with various structures, topologies and complexes with proteins are reviewed. The major emphasis is given to methods utilizing chemical functionalization of mica, enabling preparation of the surfaces with required characteristics. The methods are illustrated by examples of imaging of different DNA structures. Special attention is given to the possibility of AFM to image the dynamics of DNA at the nanoscale. The capabilities of time-lapse AFM in aqueous solutions are illustrated by imaging of dynamic processes as transitions of local alternative structures (transition of DNA between H and B forms). The application of AFM to studies of protein-DNA complexes is illustrated by a few examples of imaging site-specific complexes, as well as such systems as chromatin. The time-lapse AFM studies of protein-DNA complexes including very recent advances with the use of high-speed AFM are reviewed. PMID:20864349

Lyubchenko, Yuri L.

2010-01-01

82

Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation.  

PubMed

CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids, using an RNA-mediated interference mechanism. Interference in type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine, Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Thermobifida fusca Cas3 bound to single-stranded (ss) DNA substrate and show that it is an obligate 3'-to-5' ssDNase that preferentially accepts substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ssDNA cleavage. We demonstrate ATP coordination and conformational flexibility of the SF2-type helicase domain. Cas3 is specifically guided toward Cascade-bound target DNA by a PAM sequence, through physical interactions with both the nontarget substrate strand and the CasA protein. The sequence of recognition events ensures well-controlled DNA targeting and degradation of foreign DNA by Cascade and Cas3. PMID:25132177

Huo, Yanwu; Nam, Ki Hyun; Ding, Fang; Lee, Heejin; Wu, Lijie; Xiao, Yibei; Farchione, M Daniel; Zhou, Sharleen; Rajashankar, Kanagalaghatta; Kurinov, Igor; Zhang, Rongguang; Ke, Ailong

2014-09-01

83

Regulated degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage.  

PubMed

Chaperone-mediated autophagy (CMA) is activated in response to cellular stressors to prevent cellular proteotoxicity through selective degradation of altered proteins in lysosomes. Reduced CMA activity contributes to the decrease in proteome quality in disease and ageing. Here, we report that CMA is also upregulated in response to genotoxic insults and that declined CMA functionality leads to reduced cell survival and genomic instability. This role of CMA in genome quality control is exerted through regulated degradation of activated checkpoint kinase 1 (Chk1) by this pathway after the genotoxic insult. Nuclear accumulation of Chk1 in CMA-deficient cells compromises cell cycle progression and prolongs the time that DNA damage persists in these cells. Furthermore, blockage of CMA leads to hyperphosphorylation and destabilization of the MRN (Mre11-Rad50-Nbs1) complex, which participates in early steps of particular DNA repair pathways. We propose that CMA contributes to maintain genome stability by assuring nuclear proteostasis. PMID:25880015

Park, Caroline; Suh, Yousin; Cuervo, Ana Maria

2015-01-01

84

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

PubMed

DDB2 is a protein playing an essential role in the lesion recognition step of the global genome sub-pathway of nucleotide excision repair (GG-NER) process. Among the proteins involved in the DNA damage response, p21(CDKN1A) (p21) has been reported to participate in NER, but also to be removed by proteolytic degradation, thanks to its association with PCNA. DDB2 is involved in the CUL4-DDB1 complex mediating p21 degradation; however, the direct interaction between DDB2, p21 and PCNA has been never investigated. Here, we show that DDB2 co-localizes with PCNA and p21 at local UV-induced DNA-damage sites, and these proteins co-immunoprecipitate in the same complex. In addition, we provide evidence that p21 is not able to bind directly DDB2, but, to this end, the presence of PCNA is required. Direct physical association of recombinant DDB2 protein with PCNA is mediated by a conserved PIP-box present in the N-terminal region of DDB2. Mutation of the PIP-box resulted in the loss of protein interaction. Interestingly, the same mutation, or depletion of PCNA by RNA interference, greatly impaired DDB2 degradation induced by UV irradiation. These results indicate that DDB2 is a PCNA-binding protein, and that this association is required for DDB2 proteolytic degradation. PMID:24200966

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

2014-01-01

85

Degradable hybrid materials based on cationic acylhydrazone dynamic covalent polymers promote DNA complexation through multivalent interactions.  

PubMed

The design of smart nonviral vectors for gene delivery is of prime importance for the successful implementation of gene therapies. In particular, degradable analogues of macromolecules represent promising targets as they would combine the multivalent presentation of multiple binding units that is necessary for achieving effective complexation of therapeutic oligonucleotides with the controlled degradation of the vector that would in turn trigger drug release. Toward this end, we have designed and synthesized hybrid polyacylhydrazone-based dynamic materials that combine bis-functionalized cationic monomers with ethylene oxide containing monomers. Polymer formation was characterized by (1) H and DOSY NMR spectroscopy and was found to take place at high concentration, whereas macrocycles were predominantly formed at low concentration. HPLC monitoring of solutions of these materials in aqueous buffers at pH values ranging from 5.0 to 7.0 revealed their acid-catalyzed degradation. An ethidium bromide displacement assay and gel electrophoresis clearly demonstrated that, despite being dynamic, these materials are capable of effectively complexing dsDNA in aqueous buffer and biological serum at N/P ratios comparable to polyethyleneimine polymers. The self-assembly of dynamic covalent polymers through the incorporation of a reversible covalent bond within their main chain is therefore a promising strategy for generating degradable materials that are capable of establishing multivalent interactions and effectively complexing dsDNA in biological media. PMID:25251569

Bouillon, Camille; Paolantoni, Delphine; Rote, Jennifer C; Bessin, Yannick; Peterson, Larryn W; Dumy, Pascal; Ulrich, Sébastien

2014-11-01

86

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

87

Secondary Electron Emission as a Measure of Sample Degradation Irradiated with Electrons and UV  

NASA Astrophysics Data System (ADS)

We studied measurement of secondary electron emission (SEE) of metal and insulating materials used for satellite thermal insulation or other such purposes. The SEE yield measurement is very important for analyzing charge accumulation on satellite surfaces for a space environment because electron emission related to irradiated electrons influences the amount of surface charge. Therefore, we tried to measure the SEE yield. Furthermore, we must consider degradation phenomena of surface materials for spacecraft caused by radioactive rays. According to the degradation of materials, those SEE yields might change compared to a non-degradation sample. Therefore, we tried to measure the SEE yields of surface materials for spacecraft which are treated using the degradation process. For preparing a degradation sample, those samples were irradiated by an electron beam and UV to simulate degradation in a space environment. We take three irradiation conditions for electron beam and UV irradiation that related with GEO orbit and operating period. This report introduces measurement results of reference materials (Au, Ag, and quartz glass) and surface materials for the satellite. Furthermore, we introduce a few results of the SEE yield of the satellite materials applied degradation process simulated space environment and discuss the relationship between the SEE yields and sample degradation when irradiated by an e-beam and UV.

Miyake, Hiroaki; Nitta, Kumi; Michizono, Shinichiro; Saito, Yoshio

2009-01-01

88

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

89

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

90

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

PubMed Central

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 (R2=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

91

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

92

Study on degradation process of famotidine hydrochloride in aqueous samples  

Microsoft Academic Search

The kinetics of famotidine (FAM) transformation under the influence of various factors, important from the environmental point of view, was investigated in aqueous solutions. The degradation processes using UV, H2O2, UV\\/H2O2, H2O2\\/Fe, and UV\\/H2O2\\/Fe were studied. Direct photolysis and H2O2-assisted photolysis showed a pseudo-first-order kinetics, while the Fenton and the photo-Fenton processes fit second-order kinetics. The provided experiments proved a

Joanna Karpi?ska; Aneta Sokó?; Monika Kobeszko; Barbara Starczewska; Urszula Czy?ewska; Marta Hryniewicka

2010-01-01

93

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

94

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

95

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

96

28 CFR 28.13 - Analysis and indexing of DNA samples.  

Code of Federal Regulations, 2014 CFR

... 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

97

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

98

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

99

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.

100

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

PubMed

DNA methylation is the most common DNA modification and perhaps the best described epigenetic modification. It is believed to be important for genomic imprinting and gene regulation and has been associated with the development of diseases such as schizophrenia and some types of cancer. Neonatal dried blood spot samples, commonly known as Guthrie cards, are routinely collected worldwide to screen newborns for diseases. Some countries, including Denmark, have been storing the excess neonatal dried blood spot samples in biobanks for decades. Representing a high percentage of the population under a certain age, the neonatal dried blood spot samples are a potential alternative to collecting new samples to study diseases. As such, neonatal dried blood spot samples have previously been used for DNA genotyping studies with excellent results. However, the amount of material available for research is often limited, challenging researchers to generate the most data from a limited quantity of material. In this proof-of-principle study, we address whether two 3.2mm disks punched from a neonatal dried blood spot sample contain enough DNA for genome-wide methylome profiling, measuring 27,578 loci at the same time. We selected two subjects and carried out the following with each: 1) collected an adult whole-blood sample as reference, 2) spotted a fraction of the whole-blood sample onto a similar type of filter paper as used in the newborn screening and stored it for 3years to serve as a dried blood spot reference, and 3) identified the archived neonatal dried blood spot samples, stored for 26-28years, in the Danish Newborn Screening Biobank as a representative of the archived samples. For comparison, we used two different kits for DNA extraction. The DNA, extracted using the Extract-N-Amp Blood PCR kit, was analyzed, and no statistically significant differences were observed (P<0.001) when we compared the methylation profile of the reference whole-blood samples to the dried blood spot references. This indicates that two 3.2mm disks contain enough material for reliable methylome profiling and that storing the whole-blood sample on neonatal dried blood spot filter paper for 3years does not interfere with the outcome of the analysis. Furthermore, we compared the adult DNA methylation profile to the neonatal dried blood spot sample profile. Approximately 50 sites in the subjects were significantly (P<0.001) different in the newborn sample compared with the adult sample. Both being healthy adults and the high quality of the DNA methylation array led to the conclusion that the archived neonatal dried blood spot samples can be used for methylome profiling, despite decades of storage and DNA degradation. In conclusion, we show that reliable methylome data can be obtained from old neonatal dried blood spot samples, by using a reasonable amount of the limited resource. This further adds to the use of neonatal dried blood spot samples in genetic research and screening and paves the way for unique population-based studies of epigenetic modifications after birth. PMID:23422032

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

2013-04-01

101

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

USGS Publications Warehouse

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

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

2015-01-01

102

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

PubMed Central

Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors. Generic sampling design and terminology are proposed to standardize and clarify interpretations of eDNA-based occupancy models. PMID:25874630

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

2015-01-01

103

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

104

Ancient DNA studies: new perspectives on old samples  

PubMed Central

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

2012-01-01

105

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

PubMed

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

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

2000-11-01

106

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

PubMed Central

The methanogenic degradation of linear alkanes is a common process in oil-impacted environments. However, little is known about the key players involved in this process. Here, the hexadecane-degrading organisms in a methanogenic, hexadecane-degrading consortium designated M82 obtained from Shengli oilfield and maintained at 35°C for over 4 years, were identified by DNA-stable isotope probing with UL-13C-hexadecane, followed by density-resolved terminal restriction fragment length polymorphism (T-RFLP) analysis, cloning and phylogenetic analysis of 16S rRNA gene fragments. Compared to the fractions of the 12C treatment, the relative abundance of two phylotypes significantly increased in the heavy fractions of the 13C-hexadecane incubated microcosm. One belongs to a uncultured member of the bacterial family Syntrophaceae, which show 95–97% rRNA sequence identity with Smithella propionica, and the other is affiliated with Methanoculleus receptaculi (>99% sequence identity). The results of the present study prove the significant role of uncultured Syntrophaceae in degradation of hexadecane, probably through syntrophic interactions with hydrogenotrophic methanogens. PMID:23840866

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

2013-01-01

107

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

PubMed Central

To identify the microorganisms involved in benzene degradation, DNA-stable isotope probing (SIP) with 13C-benzene was applied to a methanogenic benzene-degrading enrichment culture. Pyrosequencing of ribosomal RNA (rRNA) gene sequences revealed that the community structure was highly complex in spite of a 3-year incubation only with benzene. The culture degraded 98% of approximately 1 mM 13C-benzene and mineralized 72% of that within 63 d. The terminal restriction fragment length polymorphism (T-RFLP) profiles of the buoyant density fractions revealed the incorporation of 13C into two phylotypes after 64 d. These two phylotypes were determined to be Desulfobacterales- and Coriobacteriaceae-related bacteria by cloning and sequencing of the 16S rRNA gene in the 13C-labeled DNA abundant fraction. Comparative pyrosequencing analysis of the buoyant density fractions of 12C- and 13C-labeled samples indicated the incorporation of 13C into three bacterial and one archaeal OTUs related to Desulfobacterales, Coriobacteriales, Rhodocyclaceae, and Methanosarcinales. The first two OTUs included the bacteria detected by T-RFLP-cloning-sequencing analysis. Furthermore, time-resolved SIP analysis confirmed that the activity of all these microbes appeared at the earliest stage of degradation. In this methanogenic culture, Desulfobacterales- and Coriobacteriaceae-related bacteria were most likely to be the major benzene degraders. PMID:24909708

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

2014-01-01

108

Importance Sampling of Word Patterns in DNA and Protein Sequences  

E-print Network

theory is absent or when either the search sequence or the word pattern is too short for the appli-specific weight matrices. 1. INTRODUCTION Searching for matches to a word pattern, also called a motifImportance Sampling of Word Patterns in DNA and Protein Sequences * HOCK PENG CHAN,1 * NANCY RUONAN

Chen, Louis H. Y.

109

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

PubMed Central

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

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

2013-01-01

110

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

111

Mutations defective in ribonucleotide reductase activity interfere with pollen plastid DNA degradation mediated by DPD1 exonuclease.  

PubMed

Organellar DNAs in mitochondria and plastids are present in multiple copies and make up a substantial proportion of total cellular DNA despite their limited genetic capacity. We recently demonstrated that organellar DNA degradation occurs during pollen maturation, mediated by the Mg(2+) -dependent organelle exonuclease DPD1. To further understand organellar DNA degradation, we characterized a distinct mutant (dpd2). In contrast to the dpd1 mutant, which retains both plastid and mitochondrial DNAs, dpd2 showed specific accumulation of plastid DNAs. Multiple abnormalities in vegetative and reproductive tissues of dpd2 were also detected. DPD2 encodes the large subunit of ribonucleotide reductase, an enzyme that functions at the rate-limiting step of de novo nucleotide biosynthesis. We demonstrated that the defects in ribonucleotide reductase indirectly compromise the activity of DPD1 nuclease in plastids, thus supporting a different regulation of organellar DNA degradation in pollen. Several lines of evidence provided here reinforce our previous conclusion that the DPD1 exonuclease plays a central role in organellar DNA degradation, functioning in DNA salvage rather than maternal inheritance during pollen development. PMID:22239102

Tang, Lay Yin; Matsushima, Ryo; Sakamoto, Wataru

2012-05-01

112

Genotyping of plant and animal samples without prior DNA purification.  

PubMed

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors. PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS) is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size. PMID:23051689

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

2012-01-01

113

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

Federal Register 2010, 2011, 2012, 2013, 2014

...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

114

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

Federal Register 2010, 2011, 2012, 2013, 2014

...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

115

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

PubMed

Investigating insulin analogs and probing their intrinsic stability at physiological temperature, we observed significant degradation in the size-exclusion chromatography (SEC) signal over a moderate number of insulin sample injections, which generated concerns about the quality of the separations. Therefore, our research goal was to identify the cause(s) for the observed signal degradation and attempt to mitigate the degradation in order to extend SEC column lifespan. In these studies, we used multiangle light scattering, nuclear magnetic resonance, and gas chromatography-mass spectrometry methods to evaluate column degradation. The results from these studies illustrate: (1) that zinc ions introduced by the insulin product produced the observed column performance issues; and (2) that including ethylenediaminetetraacetic acid, a zinc chelator, in the mobile phase helped to maintain column performance. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1555-1560, 2015. PMID:25581527

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

2015-04-01

116

DNA hydrodynamic degradation controlled by Kolomogorov length scales in pipe flow.  

PubMed

Strict US Food and Drug Administration regulations on contamination levels for DNA therapeutics acceptable for human use complicate the manufacturing process. This study aims to improve therapeutic production through the investigation of the molecular effects of hydrodynamic forces encountered during processing. Results suggest that the strain rate and residence time were not solely responsible for degradation within the system. Instead, turbulent flows at the entrance or developing flow regions dominate especially when the Kolmogorov length scale approaches the stretched molecular length scale. We specifically suggest this for linear genomic DNA and supercoiled plasmid DNA when the ratio of the molecular length to the Kolmogorov length scale must remain smaller than unity to minimize loss of the desired structure. These findings suggest that bioprocessing systems should design expansions and contractions to minimize recirculation and turbulent mixing zones, although, not always possible, careful attention should be paid to pipe surface roughness to ensure that turbulent eddies are not generated in low Reynolds number flows. PMID:21523785

Lengsfeld, Corinne S; Munson, Leslie; Lentz, Yvonne K; Anchordoquy, Thomas J

2011-08-01

117

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

USGS Publications Warehouse

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

Ferrer, I.; Barcelo, D.

2001-01-01

118

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

PubMed Central

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

2014-01-01

119

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning  

PubMed Central

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested. PMID:21445042

Diaz, Elva; Barisone, Gustavo A.

2011-01-01

120

Sensitive digital quantification of DNA methylation in clinical samples  

PubMed Central

Abnormally methylated genes are increasingly being used as cancer biomarkers 1, 2. For clinical applications, it is important to precisely determine the number of methylated molecules in the analyzed sample. We here describe a digital approach that can enumerate one methylated molecule out of ~5000 unmethylated molecules. Individual DNA fragments can be amplified and analyzed either by flow cytometry or next generation sequencing instruments. Using methylated vimentin as a biomarker, we tested 191 plasma samples and detected cancer cases with 59% sensitivity (95% CI, 48%–70%) and 93% specificity (95% CI, 86%–97%). Using the same assay, we analyzed 80 stool samples and demonstrated 45% sensitivity for detecting colorectal adenomas (23%–68%), 41% sensitivity for detecting cancer (21%–64%), and 95% specificity (82%–99%). This digital quantification of rare methylation events should be applicable to diagnostic evaluations of clinical samples, to preclinical assessments of new epigenetic biomarkers, and to quantitative analyses of epigenetic biology. PMID:19684580

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

2010-01-01

121

BIOTIC AND ABIOTIC DEGRADATION RATES OF METHYL PARATHION IN FRESHWATER AND ESTUARINE WATER AND SEDIMENT SAMPLES  

EPA Science Inventory

Statistical analysis of degradation rates of methyl parathion samples from two Gulf Coast estuaries over a three-year period indicated that biodegradation occurred in the presence of sediment but was insignificant in water. Sediment rates always showed the same relative five-fold...

122

Generation of Learning Samples for Historical Handwriting Recognition Using Image Degradation  

E-print Network

Generation of Learning Samples for Historical Handwriting Recognition Using Image Degradation handwriting recognition systems. Besides the high variabil- ity of character shapes inherent to all handwriting, the image quality can also differ greatly, for instance due to faded ink, ink bleed

Boyer, Edmond

123

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

124

Complexes of HIV-1 RT, NNRTI and RNA/DNA hybrid reveal a structure compatible with RNA degradation  

PubMed Central

Structures of type-1 human immunodeficiency virus (HIV-1) reverse transcriptase (RT) have been determined in several forms, but only one contains an RNA/DNA hybrid. Here we report three structures of HIV-1 RT complexed with a non-nucleotide RT inhibitor (NNRTI) and an RNA/DNA hybrid. In the presence of an NNRTI, the RNA/DNA structure differs from all prior nucleic acid bound to RT including the RNA/DNA hybrid. The enzyme structure also differs from all previous RT–DNA complexes. As a result, the hybrid has ready access to the RNase H active site. These observations indicate that an RT–nucleic acid complex may adopt two structural states, one competent for DNA polymerization and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites often map to the unique RT–hybrid interface that undergoes conformational changes between two catalytic states. PMID:23314251

Miller, Jennifer T.; Le Grice, Stuart F. J.; Yang, Wei

2014-01-01

125

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

126

Synthesis, spectral characterization and eukaryotic DNA degradation of thiosemicarbazones and their platinum(IV) complexes.  

PubMed

The condensation products of acetophenone (or its derivatives), salicylaldehyde and o-hydroxy-p-methoxybenzophenone with thiosemicarbazide and ethyl- or phenyl-thiosemicarbazide are the investigated thiosemicarbazones. Their reactions with H(2)PtCl(6) produced Pt(IV) complexes characterized by elemental, thermal, mass, IR and electronic spectral studies. The coordination modes were found mononegative bidentate in the acetophenone derivatives and binegative tridentate in the salicylaldehyde derivatives. The complexes were analyzed thermogravimetrically and found highly stable. Some ligands and their complexes were screened against Sarcina sp. and E. coli using the cup-diffusion technique. [Pt(oHAT)(OH)Cl] shows higher activity against E. coli than the other compounds. The degradation power of the tested compounds on the calf thymus DNA supports their selectivity against bacteria and not against the human or related eukaryotic organisms. PMID:17500028

Al-Hazmi, G A; El-Metwally, N M; El-Gammal, O A; El-Asmy, A A

2008-01-01

127

Multiple pathways are involved in DNA degradation during keratinocyte terminal differentiation  

PubMed Central

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

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

2014-01-01

128

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

Microsoft Academic Search

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

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

2010-01-01

129

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

E-print Network

1 Leishmania DNA is rapidly degraded following parasite death: An analysis by microscopy and real du Dr Roux, 75724 Paris cédex 15, France b Unité de Biologie des Interactions Hôte-Parasite of parasite burden during chemotherapy patient follow-up as well as in pharmacological studies performed

Paris-Sud XI, Université de

130

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

131

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

132

Sequencing of mtDNA HV1 and HV2 regions from samples with trace amount of DNA  

Microsoft Academic Search

Some biological samples cannot be genotyped with routine STR analyzes, if they contain trace amount of DNA. At these cases, mitochondrial DNA analyzes can be carried out. The aim of this work was optimizing the sequencing of mtDNA's HV1 and HV2 regions from samples like hair, nails, earrings, toothbrushes, q-tips, glass edge swabs, gums, razors and cigarette butts. At this

S. Erdem; H. Altunçul; G. Filo?lu; A. M. Ölçen; Ö. Bülbül

133

Microchip bioprocessor for integrated nanovolume sample purification and DNA sequencing.  

PubMed

A microfabricated electrophoretic bioprocessor for integrated DNA sequencing sample desalting, template removal, preconcentration, and CE analysis is presented. A low-viscosity gel capture matrix, containing an acrylamide-copolymerized oligonucleotide complementary to the 20-base sequence directly 3' of the M13-40 universal forward priming site, is introduced into the 60-nL capture chamber. Unpurified DNA sequencing reaction products are electrophoretically driven through the chamber; extension products hybridize to the matrix, while contaminating buffering ions, Cl-, excess primer, and template DNA are unretained. Purification under optimized conditions is complete in only 120 s (binding temperature 50 degrees C, driving voltage 250 V). High-speed, integrated sequencing analysis is accomplished by releasing the gel-purified duplex at 67 degrees C and directly injecting onto a 15.9-cm effective length CE microchannel. Electrophoretic resolution of the sequencing products is complete in 32 min, producing a total of 560 bp with phred quality q > or = 20 (accuracy > or = 99%). This fully integrated nanoliter process decreases the purification time approximately 10-fold and the process volume approximately 100-fold while providing state-of-the-art sequencing results. PMID:12380835

Paegel, Brian M; Yeung, Stephanie H I; Mathies, Richard A

2002-10-01

134

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

135

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

136

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

137

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

138

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

PubMed

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

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

2014-10-01

139

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 ...

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

140

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

141

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

142

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

143

Protocol for optimal quality and quantity pollen DNA isolation from honey samples.  

PubMed

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

2014-12-01

144

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

Microsoft Academic Search

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

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

1994-01-01

145

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

DOEpatents

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

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

1989-05-30

146

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...

147

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

148

Optimising Bacterial DNA Extraction from Faecal Samples: Comparison of Three Methods  

PubMed Central

Culture independent methods are used widely in diagnostic laboratories for infectious disease Isolation of genomic DNA from clinical samples is the first and important step in the procedure. Several procedures for extracting DNA from faecal samples have been described, including different mechanical cell disruptors. To our knowledge, the use of TissueLyser as a mechanical disruptor on faecal samples before DNA extraction has not been previously described. The purpose of the study was to implement a method for preparing faecal samples for optimal DNA extraction. Thus, three different procedures for extracting DNA from human faeces were compared. This was done either by using the mechanical disrupter by Mini BeadBeater 8, or the TissueLyser both followed by DNA purification using QIAamp DNA stool MiniKit, in comparison with DNA extractions using QIAamp DNA stool MiniKit without any prior mechanical disruption, according to manufacturer’s instructions. The obtained DNA from the three procedures was analysed by DGGE, and the number of bands was compared between each procedure. There was no significant difference between the numbers of bacterial bands obtained from DGGE when using a TissueLyser or Mini BeadBeater 8, so the two different mechanical cell disruptors can be used comparably when isolating bacterial DNA from faecal samples. The QIAamp DNA stool MiniKit alone resulted in a reduced number of bands compared to the two mechanical disruption methods. PMID:21643498

Smith, Birgitte; Li, Nan; Andersen, Anders Schou; Slotved, Hans Christian; Krogfelt, Karen Angeliki

2011-01-01

149

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

150

Resveratrol Analysis and Degradation Study in Blueberry Samples by HPLC with Fluorescence Detection  

Microsoft Academic Search

Separation and quantification of trans-resveratrol in blueberry extracts were achieved using high performance liquid chromatography (HPLC). Trans-resveratrol in blueberry juice was found to be at a high level of 16.60 ± 0.19 µg\\/mL. This antioxidant polyphenolic compound was also quantified, in two brands of blueberry powder, to be 236.4 ± 7.5 µg\\/g or 269.9 ± 5.7 µg\\/g. Degradation of trans-resveratrol was observed in standard solutions and in blueberry samples

Meera Shanmuganathan; Paul C. H. Li

2009-01-01

151

Evaluating ethanol-based sample preservation to facilitate use of DNA barcoding in routine freshwater biomonitoring programs using benthic macroinvertebrates.  

PubMed

Molecular methods, such as DNA barcoding, have the potential to 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 biomonitoring using benthic macroinvertebrates. Using higher volumes or concentrations of ethanol, requirements for shorter holding times, or the need to include additional filtering may increase cost and logistical constraints to existing biomonitoring programs. To address this issue we evaluated the efficacy of various ethanol-based sample preservation methods at maintaining DNA integrity. We evaluated a series of methods that were minimally modified from typical field protocols in order to identify an approach that can be readily incorporated into existing monitoring programs. Benthic macroinvertebrates were collected from a minimally disturbed stream in southern California, USA and subjected to one of six preservation treatments. Ten individuals from five taxa were selected from each treatment and processed to produce DNA barcodes from the mitochondrial gene cytochrome c oxidase I (COI). On average, we obtained successful COI sequences (i.e. either full or partial barcodes) for between 93-99% of all specimens across all six treatments. As long as samples were initially preserved in 95% ethanol, successful sequencing of COI barcodes was not affected by a low dilution ratio of 2?1, transfer to 70% ethanol, presence of abundant organic matter, or holding times of up to six months. Barcoding success varied by taxa, with Leptohyphidae (Ephemeroptera) producing the lowest barcode success rate, most likely due to poor PCR primer efficiency. Differential barcoding success rates have the potential to introduce spurious results. However, routine preservation methods can largely be used without adverse effects on DNA integrity. PMID:23308097

Stein, Eric D; White, Bryan P; Mazor, Raphael D; Miller, Peter E; Pilgrim, Erik M

2013-01-01

152

Bisulfite genomic sequencing of DNA from dried blood spot microvolume samples  

Microsoft Academic Search

DNA methylation is an important event in epigenetic changes in cells, and a fundamental regulator of gene transcription. Bisulfite genomic sequencing is a powerful technique used in studies of DNA methylation. However, the established procedures often require relatively large amounts of DNA. In everyday practice, samples submitted for analysis might contain very small amounts of poor quality material, as is

Hongmei Xu; Yun Zhao; Zhiping Liu; Wei Zhu; Yueqin Zhou; Ziqin Zhao

153

Herpes Simplex Virus Type 1 Immediate-Early Protein Vmw110 Induces the Proteasome-Dependent Degradation of the Catalytic Subunit of DNA-Dependent Protein Kinase  

Microsoft Academic Search

Herpes simplex virus type 1 (HSV-1) infection causes the active degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), and this process is reliant on the expression of the HSV-1 immediate-early protein Vmw110. In this study we investigated in more detail the mechanism by which the degradation occurs, the domains of Vmw110 which are required, and whether Vmw110 is

JANE PARKINSON; SUSAN P. LEES-MILLER; ROGER D. EVERETT

1999-01-01

154

High resolution melting curve analysis of DNA samples isolated by different DNA extraction methods  

Microsoft Academic Search

BackgroundHigh resolution melting is a post-PCR-based method for detecting DNA sequence variation by measuring changes in the melting of a DNA duplex. Melting of double-stranded DNA molecules is influenced by several factors. We evaluated the influence of the DNA isolation method in the melting curve analysis to detect genetic variations.

Gracia M. Martín-Núñez; Juan M. Gómez-Zumaquero; Federico Soriguer; Sonsoles Morcillo

155

Roles of ExoI and SbcCD nucleases in "reckless" DNA degradation in recA mutants of Escherichia coli.  

PubMed

Exponentially growing recA mutant cells of Escherichia coli display pronounced DNA degradation that starts at the sites of DNA damage and depends on RecBCD nuclease (ExoV) activity. As a consequence of this "reckless" DNA degradation, populations of recA mutants contain a large proportion of anucleate cells. We have found that both DNA degradation and anucleate-cell production are efficiently suppressed by mutations in the xonA (sbcB) and sbcD genes. The suppressive effects of these mutations were observed in normally grown, as well as in UV-irradiated, recA cells. The products of the xonA and sbcD genes are known to code for the ExoI and SbcCD nucleases, respectively. Since both xonA and sbcD mutations are required for strong suppression of DNA degradation while individual mutations have only a weak suppressive effect, we infer that ExoI and SbcCD play partially redundant roles in regulating DNA degradation in recA cells. We suggest that their roles might be in processing (blunting) DNA ends, thereby producing suitable substrates for RecBCD binding. PMID:19074388

Zahradka, Ksenija; Buljubasi?, Maja; Petranovi?, Mirjana; Zahradka, Davor

2009-03-01

156

Roles of ExoI and SbcCD Nucleases in “Reckless” DNA Degradation in recA Mutants of Escherichia coli?  

PubMed Central

Exponentially growing recA mutant cells of Escherichia coli display pronounced DNA degradation that starts at the sites of DNA damage and depends on RecBCD nuclease (ExoV) activity. As a consequence of this “reckless” DNA degradation, populations of recA mutants contain a large proportion of anucleate cells. We have found that both DNA degradation and anucleate-cell production are efficiently suppressed by mutations in the xonA (sbcB) and sbcD genes. The suppressive effects of these mutations were observed in normally grown, as well as in UV-irradiated, recA cells. The products of the xonA and sbcD genes are known to code for the ExoI and SbcCD nucleases, respectively. Since both xonA and sbcD mutations are required for strong suppression of DNA degradation while individual mutations have only a weak suppressive effect, we infer that ExoI and SbcCD play partially redundant roles in regulating DNA degradation in recA cells. We suggest that their roles might be in processing (blunting) DNA ends, thereby producing suitable substrates for RecBCD binding. PMID:19074388

Zahradka, Ksenija; Buljubaši?, Maja; Petranovi?, Mirjana; Zahradka, Davor

2009-01-01

157

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...

158

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

PubMed Central

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

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

2013-01-01

159

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

PubMed Central

Summary 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 are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a Protospacer Adjacent Motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R-loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg2+-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization. PMID:22521689

Westra, Edze R.; van Erp, Paul B. G.; Künne, Tim; Wong, Shi Pey; Staals, Raymond H. J.; Seegers, Christel L. C.; Bollen, Sander; Jore, Matthijs M.; Semenova, Ekaterina; Severinov, Konstantin; de Vos, Willem M.; Dame, Remus T.; de Vries, Renko; Brouns, Stan J. J.; van der Oost, John

2012-01-01

160

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

161

DNA ISOLATION FROM SMALL TISSUE SAMPLES USING SALT AND SPERMINE  

EPA Science Inventory

Common DNA isolation methods rely upon protein denaturation by organic solvents such as phenol and chloroform. hese solvents pose some risk to the user and require special disposal procedures. e have previously reported a method for isolating DNA from peripheral blood lymphocytes...

162

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

163

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

164

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

165

DNA purification from crude samples for human identification using gradient elution isotachophoresis.  

PubMed

Gradient elution isotachophoresis (GEITP) was demonstrated for DNA purification, concentration, and quantification from crude samples, represented here by soiled buccal swabs, with minimal sample preparation prior to human identification using STR analysis. During GEITP, an electric field applied across leading and trailing electrolyte solutions resulted in isotachophoretic focusing of DNA at the interface between these solutions, while a pressure-driven counterflow controlled the movement of the interface from the sample reservoir into a microfluidic capillary. This counterflow also prevented particulates from fouling or clogging the capillary and reduced or eliminated contamination of the delivered DNA by PCR inhibitors. On-line DNA quantification using laser-induced fluorescence compared favorably with quantitative PCR measurements and potentially eliminates the need for quantitative PCR prior to STR analysis. GEITP promises to address the need for a rapid and robust method to deliver DNA from crude samples to aid the forensic community in human identification. PMID:23784689

Strychalski, Elizabeth A; Konek, Christopher; Butts, Erica L R; Vallone, Peter M; Henry, Alyssa C; Ross, David

2013-09-01

166

Defects in DNA degradation revealed in crystal structures of TREX1 exonuclease mutations linked to autoimmune disease  

PubMed Central

Mutations within the human TREX1 3' exonuclease are associated with Aicardi-Goutières Syndrome (AGS) and familial chilblain lupus (FCL). Both AGS and FCL are autoimmune diseases that result in increased levels of interferon alpha and circulating antibodies to DNA. TREX1 is a member of the endoplasmic reticulum (ER)-associated SET complex and participates in granzyme A-mediated cell death to degrade nicked genomic DNA. The loss of TREX1 activity may result in the accumulation of double-stranded DNA (dsDNA) degradation intermediates that trigger autoimmune activation. The X-ray crystal structures of the TREX1 wt apoprotein, the dominant D200H, D200N and D18N homodimer mutants derived from AGS and FCL patients, as well as the recessive V201D homodimer mutant have been determined. The structures of the D200H and D200N mutant proteins reveal the enzyme has lost coordination of one of the active site metals, and the catalytic histidine (H195) is trapped in a conformation pointing away from the active site. The TREX1 D18N and V201D mutants are able to bind both metals in the active site, but with inter-metal distances that are larger than optimal for catalysis. Additionally, all of the mutant structures reveal a reduced mobility in the catalytic histidine, providing further explanation for the loss of catalytic activity. The structures of the mutant TREX1 proteins provide insight into the dysfunction relating to human disease. Additionally, the TREX1 apoprotein structure together with the previously determined wild type substrate and product structures allow us to propose a distinct mechanism for the TREX1 exonuclease. PMID:22071149

Bailey, Suzanna L.; Harvey, Scott; Perrino, Fred W.; Hollis, Thomas

2011-01-01

167

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

PubMed Central

HIV-1 reverse transcriptase discriminates poorly between dUTP and dTTP, and accordingly, viral DNA products become heavily uracilated when viruses infect host cells that contain high ratios of dUTP:dTTP. Uracilation of invading retroviral DNA is thought to be an innate immunity barrier to retroviral infection, but the mechanistic features of this immune pathway and the cellular fate of uracilated retroviral DNA products is not known. Here we developed a model system in which the cellular dUTP:dTTP ratio can be pharmacologically increased to favor dUTP incorporation, allowing dissection of this innate immunity pathway. When the virus-infected cells contained elevated dUTP levels, reverse transcription was found to proceed unperturbed, but integration and viral protein expression were largely blocked. Furthermore, successfully integrated proviruses lacked detectable uracil, suggesting that only nonuracilated viral DNA products were integration competent. Integration of the uracilated proviruses was restored using an isogenic cell line that had no detectable human uracil DNA glycosylase (hUNG2) activity, establishing that hUNG2 is a host restriction factor in cells that contain high dUTP. Biochemical studies in primary cells established that this immune pathway is not operative in CD4+ T cells, because these cells have high dUTPase activity (low dUTP), and only modest levels of hUNG activity. Although monocyte-derived macrophages have high dUTP levels, these cells have low hUNG activity, which may diminish the effectiveness of this restriction pathway. These findings establish the essential elements of this pathway and reconcile diverse observations in the literature. PMID:23341616

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

2013-01-01

168

Exploring Hidden Genetic Divergence Within Sunda Colugos by Means of Novel DNA Capture Methods and Next Generation Sequencing  

E-print Network

colugo (Galeopterus variegatus) to redefine the evolutionary relationships between disjunct populations of this poorly understood mammal, using a novel DNA capture method to isolate degraded mtDNA fragments from museum samples, by hybridization to DNA...

Mason, Victor C

2012-07-11

169

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

170

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

NASA Astrophysics Data System (ADS)

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

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

2006-05-01

171

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

PubMed

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

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

2006-05-30

172

Nucleotide-Dependent Degradation of Nucleic Acids by DNA and RNA Polymerases  

Microsoft Academic Search

The review considers two reactions discovered recently: (1) excision of the 3'-terminal nucleotide from the nascent DNA strand in the presence of relatively high concentrations of noncomplementary r\\/dNTPs or r\\/dNDPs (pseudopyrophosphorolysis), which is catalyzed by various DNA polymerases (DNAPs), and (2) excision of the 3'-terminal nucleotide from the nascent RNA strand, which is catalyzed by RNA polymerases (RNAPs) and stimulated

V. V. Sosunov; L. S. Victorova

2004-01-01

173

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

PubMed

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 measures. The methods included solid-phase DNA adsorption (QIAamp), sequential protein and DNA precipitation (Puregene), magnetic bead adsorption (Dynabeads), phenol-chloroform extraction, and single-step proteinase K digestion. In general, the performance of the three commercial kits was superior to either phenol-chloroform extraction or single-step proteinase K digestion. However, QIAamp and Puregene produced amplifiable DNA more frequently than Dynabeads for starting cell numbers <50,000. GAPDH Ct values for QIAamp extractions showed the greatest dynamic range and the best linearity across the range of starting cell numbers, but QIAamp was not statistically significantly superior to Puregene. Of the three commercial kits, Puregene is the least expensive. QIAamp and Puregene DNA extraction methods are well-suited for the preparation of paucicellular clinical samples for PCR-based assays. PMID:16516438

Cler, Leslie; Bu, Dawei; Lewis, Cheryl; Euhus, David

2006-01-01

174

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

Microsoft Academic Search

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

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

2011-01-01

175

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

E-print Network

To Clone or Not To Clone: Method Analysis for Retrieving Consensus Sequences In Ancient DNA Samples methods considered to be ``gold standards'' in the field, including cloning aDNA amplicons as opposed of clones to sequence, how a consensus sequence is most appropriately derived, or how results should

Kemp, Brian M.

176

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

177

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

PubMed

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

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

2001-12-01

178

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

E-print Network

ORIGINAL PAPER Evaluating the impact of non-lethal DNA sampling on two butterflies, Vanessa cardui of Vanessa cardui and Satyrodes eurydice. Based on these studies we were successful in obtaining a permit

Landis, Doug

179

Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples  

E-print Network

Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples Center for Environmental Health, Institute of Groundwater Ecology, Neuherberg, Germany. Summary., 2004; Sullivan et al., 2006), phosphate stress response (Sullivan et al., 2005), nitrogen stress

Sullivan, Matthew B.

180

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

PubMed Central

Background Technical advances following the Human Genome Project revealed that high-quality and -quantity DNA may be obtained from whole saliva samples. However, usability of previously collected samples and the effects of environmental conditions on the samples during collection have not been assessed in detail. In five studies we document the effects of sample volume, handling and storage conditions, type of collection device, and oral sampling location, on quantity, quality, and genetic assessment of DNA extracted from cells present in saliva. Methods Saliva samples were collected from ten adults in each study. Saliva volumes from .10-1.0 ml, different saliva collection devices, sampling locations in the mouth, room temperature storage, and multiple freeze-thaw cycles were tested. One representative single nucleotide polymorphism (SNP) in the catechol-0-methyltransferase gene (COMT rs4680) and one representative variable number of tandem repeats (VNTR) in the serotonin transporter gene (5-HTTLPR: serotonin transporter linked polymorphic region) were selected for genetic analyses. Results The smallest tested whole saliva volume of .10 ml yielded, on average, 1.43 ± .77 ?g DNA and gave accurate genotype calls in both genetic analyses. The usage of collection devices reduced the amount of DNA extracted from the saliva filtrates compared to the whole saliva sample, as 54-92% of the DNA was retained on the device. An "adhered cell" extraction enabled recovery of this DNA and provided good quality and quantity DNA. The DNA from both the saliva filtrates and the adhered cell recovery provided accurate genotype calls. The effects of storage at room temperature (up to 5 days), repeated freeze-thaw cycles (up to 6 cycles), and oral sampling location on DNA extraction and on genetic analysis from saliva were negligible. Conclusions Whole saliva samples with volumes of at least .10 ml were sufficient to extract good quality and quantity DNA. Using 10 ng of DNA per genotyping reaction, the obtained samples can be used for more than one hundred candidate gene assays. When saliva is collected with an absorbent device, most of the nucleic acid content remains in the device, therefore it is advisable to collect the device separately for later genetic analyses. PMID:22182470

2011-01-01

181

Degradation of mitochondrial DNA in cryoprotectant-treated hard coral (Echinopora spp.) oocytes.  

PubMed

Abstract A critical step for successful cryopreservation is to determine the optimal cryoprotectant treatment that can provide protective effects against cryoinjury during freezing and with minimal toxicity. Most cryoprotectants have chemical and osmotic effects when used at high concentrations. Cryoprotectants can damage coral mitochondrial distributions and membrane potentials, which results in reduced ATP production. As mitochondrial DNA (mtDNA) encodes for components of the electron transport chain (ETC) and plays a critical role in ATP synthesis capacity, we determined the effects of cryoprotectants on mtDNA in hard coral (Echinopora spp.) oocytes using quantitative real-time PCR. Our results showed that an insult from a cryoprotectant may be compensated for by the genetic defense mechanisms of these cells. Methanol was found to have the least effect on coral oocytes with regard to their energy status. A single oocyte without cryoprotectant treatment produced an average of 4,220,645?±?169,990 mtDNA copies, which was greater than that in mammals. However, relatively lower mtDNA copy numbers (<2,000,000) were observed when oocytes were treated with dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), or glycerol at a concentration of 3?M for 20?min. These results provide direct evidence that hard coral (Echinopora spp.) oocytes are extremely susceptible to cryoprotectants and support the concerns with regard to the adverse effects of cryoprotectants. PMID:24460160

Tsai, Sujune; Chen, Jiann-Chu; Spikings, Emma; Li, Jan-Jung; Lin, Chiahsin

2014-01-27

182

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

1989-01-01

183

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

184

A comparison of molecular methods for hepatitis B virus (HBV) DNA detection from oral fluid samples.  

PubMed

The objective of the present study was to evaluate four commercial DNA extraction methods and three PCR protocols for hepatitis B virus (HBV) detection in artificially contaminated oral fluid samples. The extraction protocols were selected based on ease of use and cost, and were also compared with respect to sensitivity and cost. Prior PCR optimization was conducted, in which the sample volume for DNA extraction and the concentrations of DNA and Taq DNA polymerase in the PCR were adjusted. One-round PCR, used to amplify the core region of the HBV genome, achieved high levels of sensitivity in comparison with nested and semi-nested PCR experiments that were designed for the amplification of HBV surface protein genes. Of the four extraction protocols evaluated, the RTP DNA/RNA Virus Mini kit and the QIAamp DNA Mini kit gave the highest recovery rates, presenting 20 copies of HBV DNA ml(-1) as the limit of detection. These results suggest that HBV DNA can be detected from oral fluid samples but that the optimization of the PCR assays and the choice of extraction methods must be determined by laboratories before the implementation of this method in routine diagnostics. PMID:22403138

Portilho, Moyra Machado; Martins, Patrícia Pais; Lampe, Elisabeth; Villar, Livia Melo

2012-06-01

185

A New Blood Collection Device Minimizes Cellular DNA Release During Sample Storage and Shipping When Compared to a Standard Device  

PubMed Central

Background Cell-free DNA (cfDNA) circulating in blood is currently used for noninvasive diagnostic and prognostic tests. Minimizing background DNA is vital for detection of low abundance cfDNA. We investigated whether a new blood collection device could reduce background levels of genomic DNA (gDNA) in plasma compared to K3EDTA tubes, when subjected to conditions that may occur during sample storage and shipping. Methods Blood samples were drawn from healthy donors into K3EDTA and Cell-Free DNA™ BCT (BCT). To simulate shipping, samples were shaken or left unshaken. In a shipping study, samples were shipped or not shipped. To assess temperature variations, samples were incubated at 6°C, 22°C, and 37°C. In all cases, plasma was harvested by centrifugation and total plasma DNA (pDNA) assayed by quantitative real-time polymerase chain reaction (qPCR). Results Shaking and shipping blood in K3EDTA tubes showed significant increases in pDNA, whereas no change was seen in BCTs. Blood in K3EDTA tubes incubated at 6°C, 22°C, and 37°C showed increases in pDNA while pDNA from BCTs remained stable. Conclusions BCTs prevent increases in gDNA levels that can occur during sample storage and shipping. This new device permits low abundance DNA target detection and allows accurate cfDNA concentrations. PMID:23852790

Norton, Sheila E; Luna, Kristin K; Lechner, Joel M; Qin, Jianbing; Fernando, M Rohan

2013-01-01

186

Ultrasound assessment of articular cartilage: analysis of the frequency profile of reflected signals from naturally and artificially degraded samples.  

PubMed

This article investigates in vitro the hypothesis that the frequency profile of ultrasound reflections may be used to characterize degradation and osteoarthritic progression in articular cartilage, irrespective of the effects of transducer orientation. To this end, ultrasound echoes were taken in the time domain from the articular surface and osteochondral junction of normal, collagen meshwork-disrupted, proteoglycan-depleted, and osteoarthritic samples, converted to the frequency domain by fast Fourier transform and analyzed. Our results show the significant effects of specific enzymatic degradation programs on the ultrasound frequency profile of reflections from the cartilage surface and osteochondral junction, and their manifestation in the tissue surrounding a focal osteoarthritic defect. Collagen meshwork disruption was most apparent in the profile of reflections from the articular surface, while proteoglycan depletion was most clearly observed in the reflections from the osteochondral junction. The reflected signals from the osteochondral junction may further contain information about the subchondral bone. From these results we proposed that the analysis of specific frequencies of reflected ultrasound signals has the potential to differentiate normal from degraded articular cartilage-on-bone, when the angle of incidence can be controlled within a +/-1.2 degrees limit. This encourages further research into the effects of progressive artificial degradation of the cartilage matrix and subchondral bone on the spectral profile to quantify the relationship between the frequency profile and the level of specific degradation in naturally degraded joints. PMID:18075813

Brown, Cameron P; Hughes, Stephen W; Crawford, Ross W; Oloyede, Adekunle

2007-01-01

187

Phylogenetic estimation of timescales using ancient DNA: the effects of temporal sampling scheme and uncertainty in sample ages.  

PubMed

In recent years, ancient DNA has increasingly been used for estimating molecular timescales, particularly in studies of substitution rates and demographic histories. Molecular clocks can be calibrated using temporal information from ancient DNA sequences. This information comes from the ages of the ancient samples, which can be estimated by radiocarbon dating the source material or by dating the layers in which the material was deposited. Both methods involve sources of uncertainty. The performance of bayesian phylogenetic inference depends on the information content of the data set, which includes variation in the DNA sequences and the structure of the sample ages. Various sources of estimation error can reduce our ability to estimate rates and timescales accurately and precisely. We investigated the impact of sample-dating uncertainties on the estimation of evolutionary timescale parameters using the software BEAST. Our analyses involved 11 published data sets and focused on estimates of substitution rate and root age. We show that, provided that samples have been accurately dated and have a broad temporal span, it might be unnecessary to account for sample-dating uncertainty in Bayesian phylogenetic analyses of ancient DNA. We also investigated the sample size and temporal span of the ancient DNA sequences needed to estimate phylogenetic timescales reliably. Our results show that the range of sample ages plays a crucial role in determining the quality of the results but that accurate and precise phylogenetic estimates of timescales can be made even with only a few ancient sequences. These findings have important practical consequences for studies of molecular rates, timescales, and population dynamics. PMID:23024187

Molak, Martyna; Lorenzen, Eline D; Shapiro, Beth; Ho, Simon Y W

2013-02-01

188

Stool sample storage conditions for the preservation of Giardia intestinalis DNA.  

PubMed

Stool is chemically complex and the extraction of DNA from stool samples is extremely difficult. Haemoglobin breakdown products, such as bilirubin, bile acids and mineral ions, that are present in the stool samples, can inhibit DNA amplification and cause molecular assays to produce false-negative results. Therefore, stool storage conditions are highly important for the diagnosis of intestinal parasites and other microorganisms through molecular approaches. In the current study, stool samples that were positive for Giardia intestinalis were collected from five different patients. Each sample was stored using one out of six different storage conditions [room temperature (RT), +4ºC, -20ºC, 70% alcohol, 10% formaldehyde or 2.5% potassium dichromate] for DNA extraction procedures at one, two, three and four weeks. A modified QIAamp Stool Mini Kit procedure was used to isolate the DNA from stored samples. After DNA isolation, polymerase chain reaction (PCR) amplification was performed using primers that target the ?-giardin gene. A G. intestinalis-specific 384 bp band was obtained from all of the cyst-containing stool samples that were stored at RT, +4ºC and -20ºC and in 70% alcohol and 2.5% potassium dichromate; however, this band was not produced by samples that had been stored in 10% formaldehyde. Moreover, for the stool samples containing trophozoites, the same G. intestinalis-specific band was only obtained from the samples that were stored in 2.5% potassium dichromate for up to one month. As a result, it appears evident that the most suitable storage condition for stool samples to permit the isolation of G. intestinalis DNA is in 2.5% potassium dichromate; under these conditions, stool samples may be stored for one month. PMID:23295744

Kuk, Salih; Yazar, Suleyman; Cetinkaya, Ulfet

2012-12-01

189

Autosomal and Y-STR analysis of degraded DNA from the 120-year-old skeletal remains of Ezekiel Harper.  

PubMed

The 120-year-old skeletal remains of Confederate Civil War soldier Captain Ezekiel "Zeke" Harper were exhumed by court order in January 2011 for DNA analysis. The goal of the DNA testing was to support or refute whether Captain Harper had fathered a son (Earl J. Maxwell) with his Native American maid prior to his murder in 1892. Bones with adequate structural integrity (left tibia, right tibia, right femur, mandible, four teeth) were retrieved from the burial site and sent to the Institute of Applied Genetics in Fort Worth, Texas for analysis. Given the age and condition of the remains, three different extraction methods were used to maximize the probability of DNA recovery. The majority of the DNA isolates from over fifty separate bone sections yielded partial autosomal STR genotypes and partial Y-STR haplotypes. After comparing the partial results for concordance, consensus profiles were generated for comparison to reference samples from alleged family members. Considering the genetic recombination that occurs in autosomal DNA over the generations within a family, Y-STR analysis was determined to be the most appropriate and informative approach for determining potential kinship. Two of Earl J. Maxwell's grandsons submitted buccal samples for comparison. The Y-STR haplotypes obtained from both of these reference samples were identical to each other and to the alleles in Ezekiel Harper's consensus profile at all 17 loci examined. This Y-STR haplotype was not found in either of two major Y-STR population databases (U.S. Y-STR database and YHRD). The fact that the Y-STR haplotype obtained from Ezekiel's skeletal remains and Earl's grandsons is not found in either population database demonstrates its rarity and further supports a paternal lineage relationship among them. Results of the genetic analyses are consistent with the hypothesis that Earl J. Maxwell is the son of Ezekiel Harper. PMID:24528577

Ambers, Angie; Gill-King, Harrell; Dirkmaat, Dennis; Benjamin, Robert; King, Jonathan; Budowle, Bruce

2014-03-01

190

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

191

Qualification Study of Two Genomic DNA Extraction Methods in Different Clinical Samples  

PubMed Central

Introduction The purity of genomic DNA (gDNA) extracted from different clinical specimens optimizes sensitivity of polymerase chain reaction (PCR) assays. This study attempted to compare two different DNA extraction techniques namely salting-out and classic phenol-chloroform. Materials and Methods Qualification of two different DNA extraction techniques for 634 clinical specimens highly suspected of having mycobacterial infection was performed. Genomic DNA was extracted from 330 clinical samples using phenol-chloroform and 304 by non-toxic salting-out. Qualification of obtained gDNA was done through amplification of internal controls, ?-actin and ?-globin. Results ?-actin-positive was detected in 279/330 (84%) and 272/304 (89%) samples by phenol-chloroform technique and salting-out, respectively. PCR inhibitor was found for the gDNA of 13/304 (4%) patient samples were negative by ?-actin and ?-globin tests via salting-out technique in comparison with gDNAs from 27/330 (8.5%) samples extracted by phenol-chloroform procedure. No statistically significant difference was found between phenol-chloroform technique and salting-out for 385 sputum, 29 bronchoalveolar lavage (BAL), 105 gastric washing, and 38 body fluid (P=0.04) samples. This illustrates that both techniques have the same quality for extracting gDNA. Conclusion This study discloses salting-out as a non-toxic DNA extraction procedure with a superior time-efficiency and cost-effectiveness in comparison with phenol-chloroform and it can be routinely used in resource-limited laboratory settings.

Javadi, Alireza; Shamaei, Masoud; Pourabdollah, Mihan; Dorudinia, Atosa; Seyedmehdi, Seyed Mohammad; Karimi, Shirin

2014-01-01

192

Sampling of herpes zoster skin lesion types and the impact on viral DNA detection.  

PubMed

This was a multicenter, non-therapeutic study to determine the optimal type of lesion sample for quantitative PCR detection of varicella zoster virus (VZV) DNA in herpes zoster patients. Up to three crusts, three crust swabs, three vesicle swabs, and three papule swabs were collected from 41 adults with clinically diagnosed herpes zoster. 83% of subjects had at least one valid crust swab (detectable VZV or ?-actin DNA), 78% had at least one valid crust, 78% had at least one valid vesicle swab, and 32% had at least one valid papule swab. Of valid samples, 97% of crusts, 94% of vesicle swabs, 90% of crust swabs, and 84% of papule swabs were VZV-DNA-positive (?10 DNA copies/sample). 37 (90%) subjects had at least one VZV DNA-positive sample. VZV DNA copy numbers were highest for vesicle swabs and crusts. The probability of a false-negative result was 5% for crusts, 6% for vesicle swabs, 14% for papule swabs, and 24% for crust swabs. Overall, vesicle swabs and crusts were the most specific and sensitive samples for detecting VZV. PMID:23275023

Mols, Johann F; Ledent, Edouard; Heineman, Thomas C

2013-03-01

193

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

PubMed Central

Background Strongyloidiasis is mostly an asymptomatic infection and diagnosis of latent infections is difficult due to limitations of current parasitological and serological methods. This study was conducted to set up a PCR-based method for molecular diagnosis of Strongyloides stercoralis infection by detection of copro-DNA in stool samples. Methods A total of 782 fresh stool samples were collected and examined by agar plate culture. Among those sixteen stool samples, which confirmed to be infected with S. stercoralis were examined as positive control to set up each single and nested PCR, using two primer sets designing to amplify partial ribosomal DNA of S. stercoralis genome. Since, single PCR method yielded higher efficacy in detecting positive samples, in the second step, 30 stool samples, which found negative for S. stercoralis by agar plate culture of single stool sample, were examined by single PCR. Data analysis was performed using McNemar's ?2 test, with consideration of a P-value of <0.05 as indication of significant difference. Results In amplification of DNA extracted from stool samples, single PCR detected S. stercoralis DNA target in all 16 positive samples, while nested PCR amplified DNA in only 75% of samples. In the second step, single PCR amplified S. stercoralis extracted DNA in 5 out of 30 samples which were negative by coproculture. Conclusion Single PCR method amplifying a short (100bp) target represented more efficacies for detection of S. stercoralis in faecal examination compared to agar plate culture and nested PCR, which amplified longer target. PMID:22347284

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

2011-01-01

194

A Papillomavirus DNA from a Cervical Carcinoma and Its Prevalence in Cancer Biopsy Samples from Different Geographic Regions  

Microsoft Academic Search

DNA from one biopsy sample of invasive cancer of the cervix contained sequences hybridizing with human papillomavirus (HPV) type 11 DNA only under nonstringent conditions. This DNA was molecularly cloned in lambda phage. Under stringent conditions of hybridization it cross-hybridized to a minor extent (less than 0.1%) with HPV types 10, 14, and 15 and showed no homology with DNA

Matthias Durst; Lutz Gissmann; Hans Ikenberg; Harald Zur Hausen

1983-01-01

195

Evaluation of human and microbial DNA content in subgingival plaque samples collected by paper points or curette.  

PubMed

Host DNA may adversely affect metagenomic studies focusing on the prokaryotic microbiota. This study compared the levels of host DNA in subgingival plaque collected by paper points and curette, using quantitative PCR. Lower proportions of host DNA and higher proportions of bacterial DNA were recovered from samples collected with curettes. PMID:25644890

Pérez-Chaparro, P J; Duarte, P M; Pannuti, C M; Figueiredo, L C; Mestnik, M J; Gonçalves, C P S; Faveri, M; Feres, M

2015-04-01

196

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

197

Phosphorylation regulates proteasomal-mediated degradation and solubility of TAR DNA binding protein-43 C-terminal fragments  

PubMed Central

Background Inclusions of TAR DNA binding protein-43 (TDP-43) are the defining histopathological feature of several neurodegenerative diseases collectively referred to as TDP-43 proteinopathies. These diseases are characterized by the presence of cellular aggregates composed of abnormally phosphorylated, N-terminally truncated and ubiquitinated TDP-43 in the spinal cord and/or brain. Recent studies indicate that C-terminal fragments of TDP-43 are aggregation-prone and induce cytotoxicity. However, little is known regarding the pathways responsible for the degradation of these fragments and how their phosphorylation contributes to the pathogenesis of disease. Results Herein, we established a human neuroblastoma cell line (M17D3) that conditionally expresses an enhanced green fluorescent protein (GFP)-tagged caspase-cleaved C-terminal TDP-43 fragment (GFP-TDP220-414). We report that expression of this fragment within cells leads to a time-dependent formation of inclusions that are immunoreactive for both ubiquitin and phosphorylated TDP-43, thus recapitulating pathological hallmarks of TDP-43 proteinopathies. Phosphorylation of GFP-TDP220-414 renders it resistant to degradation and enhances its accumulation into insoluble aggregates. Nonetheless, GFP-TDP220-414 inclusions are reversible and can be cleared through the ubiquitin proteasome system. Moreover, both Hsp70 and Hsp90 bind to GFP-TDP220-414 and regulate its degradation. Conclusions Our data indicates that inclusions formed from TDP-43 C-terminal fragments are reversible. Given that TDP-43 inclusions have been shown to confer toxicity, our findings have important therapeutic implications and suggest that modulating the phosphorylation state of TDP-43 C-terminal fragments may be a promising therapeutic strategy to clear TDP-43 inclusions. PMID:20804554

2010-01-01

198

Bisulfite genomic sequencing of DNA from dried blood spot microvolume samples.  

PubMed

DNA methylation is an important event in epigenetic changes in cells, and a fundamental regulator of gene transcription. Bisulfite genomic sequencing is a powerful technique used in studies of DNA methylation. However, the established procedures often require relatively large amounts of DNA. In everyday practice, samples submitted for analysis might contain very small amounts of poor quality material, as is often the case with forensic stain samples. In this study, we assess a modified, more efficient method of bisulfite genomic sequencing. Genomic DNA extracted from 3-mm dried blood spots using QIAamp micro kit was treated with sodium bisulfite (using EpiTect kit). Subsequent methylation-specific PCR (MSP) followed by DNA sequencing displayed the differentially methylated region of imprinted gene SNRPN. Our results show that this new combination of efficient DNA extraction and bisulfite treatment provides high quality conversion of unmethylated cytosine to uracil for bisulfite genomic sequencing analysis. This reliable method substantially improves the DNA methylation analysis of forensic stain samples. PMID:21737370

Xu, Hongmei; Zhao, Yun; Liu, Zhiping; Zhu, Wei; Zhou, Yueqin; Zhao, Ziqin

2012-05-01

199

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

200

PNA microarrays for hybridisation of unlabelled DNA samples  

PubMed Central

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

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

2003-01-01

201

Estimating the Age of the Common Ancestor of a Sample of DNA Sequences  

Microsoft Academic Search

We present a simple Monte Carlo method for estimating the age of the most recent common ancestor (MRCA) of a sample of DNA sequences. We show that Templeton's (1993) estimator of the age of the MRCA based on the maximum number of nucleotide differences between two sequences in a sample is inaccurate, and we demonstrate the new method by reanalyzing

Yun-Xin Fu; Wen-Hsiung Li

202

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

Microsoft Academic Search

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

Howard B. Lieberman; Evelyn M. Witkin

1983-01-01

203

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

204

RNF8- and RNF168-dependent degradation of KDM4A/JMJD2A triggers 53BP1 recruitment to DNA damage sites  

PubMed Central

In response to DNA damage, cells initiate complex signalling cascades leading to growth arrest and DNA repair. The recruitment of 53BP1 to damaged sites requires the activation of the ubiquitination cascade controlled by the E3 ubiquitin ligases RNF8 and RNF168, and methylation of histone H4 on lysine 20. However, molecular events that regulate the accessibility of methylated histones, to allow the recruitment of 53BP1 to DNA breaks, are unclear. Here, we show that like 53BP1, the JMJD2A (also known as KDM4A) tandem tudor domain binds dimethylated histone H4K20; however, JMJD2A is degraded by the proteasome following the DNA damage in an RNF8-dependent manner. We demonstrate that JMJD2A is ubiquitinated by RNF8 and RNF168. Moreover, ectopic expression of JMJD2A abrogates 53BP1 recruitment to DNA damage sites, indicating a role in antagonizing 53BP1 for methylated histone marks. The combined knockdown of JMJD2A and JMJD2B significantly rescued the ability of RNF8- and RNF168-deficient cells to form 53BP1 foci. We propose that the RNF8-dependent degradation of JMJD2A regulates DNA repair by controlling the recruitment of 53BP1 at DNA damage sites. PMID:22373579

Mallette, Frédérick A; Mattiroli, Francesca; Cui, Gaofeng; Young, Leah C; Hendzel, Michael J; Mer, Georges; Sixma, Titia K; Richard, Stéphane

2012-01-01

205

The ?2 helix in the DNA Ligase IV BRCT-1 domain is required for targeted degradation of Ligase IV during adenovirus infection  

PubMed Central

In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood. Through genetic analysis, we identified the ?2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 ?2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 ?2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV. PMID:22534089

Gilson, Timra; Greer, Amy E.; Vindigni, Alessandro; Ketner, Gary; Hanakahi, Leslyn A.

2012-01-01

206

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

Microsoft Academic Search

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

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

2002-01-01

207

Requirement of DNA Repair Mechanisms for Survival of Burkholderia cepacia G4 upon Degradation of Trichloroethylene  

PubMed Central

A Tn5-based mutagenesis strategy was used to generate a collection of trichloroethylene (TCE)-sensitive (TCS) mutants in order to identify repair systems or protective mechanisms that shield Burkholderia cepacia G4 from the toxic effects associated with TCE oxidation. Single Tn5 insertion sites were mapped within open reading frames putatively encoding enzymes involved in DNA repair (UvrB, RuvB, RecA, and RecG) in 7 of the 11 TCS strains obtained (4 of the TCS strains had a single Tn5 insertion within a uvrB homolog). The data revealed that the uvrB-disrupted strains were exceptionally susceptible to killing by TCE oxidation, followed by the recA strain, while the ruvB and recG strains were just slightly more sensitive to TCE than the wild type. The uvrB and recA strains were also extremely sensitive to UV light and, to a lesser extent, to exposure to mitomycin C and H2O2. The data from this study establishes that there is a link between DNA repair and the ability of B. cepacia G4 cells to survive following TCE transformation. A possible role for nucleotide excision repair and recombination repair activities in TCE-damaged cells is discussed. PMID:11722883

Yeager, Chris M.; Bottomley, Peter J.; Arp, Daniel J.

2001-01-01

208

Efficient removal of DNA from proteomic samples prior to two-dimensional map analysis.  

PubMed

Several methods have been described in the literature for removal of DNA from protein samples prior to proteome analysis. They in general involve protein precipitation techniques. In other protocols, DNAse treatment is suggested prior to precipitation of proteins in excess acetone. All these methods have been evaluated and found to perform poorly in DNA removal, as illustrated by two-dimensional (2D) maps where horizontal and vertical sample streaking are still substantial. Such removal is in general necessary in tissue lysates and especially when analysing sub-cellular organelles, such as nuclei, where the high DNA levels strongly interfere with proteome analysis. Another method is proposed here for efficient DNA removal: two-phase extraction of DNA in chloroform/phenol/isoamyl alcohol, a procedure commonly used to rid DNA samples of protein contaminants, but rarely applied to protein preparation. This extraction is not very efficient if performed at slightly acidic to neutral pH values, but it performs extremely well at pH values of 9.5 or higher. The 2D maps thus obtained of Escherichia coli lysates as well as extracts from purified nuclei of eukaryotic cells are not only devoid of any vertical or horizontal streaking, but exhibit many more spots, especially in the alkaline region of the 2D gels, suggesting that these basic proteins were in general lost to proteome analysis due to co-precipitation in tenacious protein-DNA complexes. It is hypothesized that the alkaline pH values adopted in the two-phase extraction help to fully disrupt any residual DNA-protein complexes, due to strong Coulombic repulsion. PMID:19081104

Antonioli, Paolo; Bachi, Angela; Fasoli, Elisa; Righetti, Pier Giorgio

2009-04-24

209

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

210

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

PubMed

An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples. PMID:15453677

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

2004-10-01

211

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

212

DNA barcoding meets molecular scatology: short mtDNA sequences for standardized species assignment of carnivore noninvasive samples.  

PubMed

Although species assignment of scats is important to study carnivore biology, there is still no standardized assay for the identification of carnivores worldwide, which would allow large-scale routine assessments and reliable cross-comparison of results. Here, we evaluate the potential of two short mtDNA fragments [ATP6 (126 bp) and cytochrome oxidase I gene (COI) (187 bp)] to serve as standard markers for the Carnivora. Samples of 66 species were sequenced for one or both of these segments. Alignments were complemented with archival sequences and analysed with three approaches (tree-based, distance-based and character-based). Intraspecific genetic distances were generally lower than between-species distances, resulting in diagnosable clusters for 86% (ATP6) and 85% (COI) of the species. Notable exceptions were recently diverged species, most of which could still be identified using diagnostic characters and uniqueness of haplotypes or by reducing the geographic scope of the comparison. In silico analyses were also performed for a 110-bp cytochrome b (cytb) segment, whose identification success was lower (70%), possibly due to the smaller number of informative sites and/or the influence of misidentified sequences obtained from GenBank. Finally, we performed case studies with faecal samples, which supported the suitability of our two focal markers for poor-quality DNA and allowed an assessment of prey DNA co-amplification. No evidence of prey DNA contamination was found for ATP6, while some cases were observed for COI and subsequently eliminated by the design of more specific primers. Overall, our results indicate that these segments hold good potential as standard markers for accurate species-level identification in the Carnivora. PMID:21883979

Chaves, Paulo B; Graeff, Vanessa G; Lion, Marília B; Oliveira, Larissa R; Eizirik, Eduardo

2012-01-01

213

Degradation products of Tenax TA formed during sampling and thermal desorption analysis: Indicators of reactive species indoors  

NASA Astrophysics Data System (ADS)

Analyses of indoor air samples of semivolatile organic compounds (SVOCs) from five offices in two office buildings, a school classroom, and a room in a day-care center were generally strongly influenced by artifact formation. In the laboratory, the major artifacts could be produced by sampling mixtures of O 3, NO 2, and limonene in air on the sorbent, Tenax TA. Several SVOCs from O 3 degradation of Tenax TA were detected, but only few were identified. The NO 2 degradation of Tenax TA analyzed by thermal desorption and gas chromatography (TD-GC) almost exclusively formed 2,6-diphenyl- p-benzoquinone (DPQ) and 2,6-diphenyl- p-hydroquinone (DPHQ). The NO 2/Tenax TA reaction could be calibrated, thus the NO 2 concentration could be determined simultaneously with a SVOC measurement. However, the results indicated that DPQ may be reduced to DPHQ during TD-GC analysis by oxidation of other compounds adsorbed to Tenax TA. Sampling an air mixture of O 3 in excess of limonene on Tenax TA followed by TD-GC analysis exclusively produced DPHQ. O 3 alone produced neither DPQ nor DPHQ. It was found that reactive species (possibly Criegee biradicals and/or other organic radicals) from the O 3/limonene :reaction were responsible for the production of DPHQ from Tenax TA. The results indicated that Tenax TA can be used as a trapping agent for some radicals by analysis of the DPQ/DPHQ formation. The present data were not sufficient to obtain evidence for degradation of Tenax TA by other radicals than NO and NO 2 in indoor SVOC samples. However, the DPQ/DPHQ ratio indicated that DPHQ has been formed from DPQ by oxidation of other adsorbed compounds in some of the samples.

Axel Clausen, Per; Wolkoff, Peder

214

An efficient and sensitive method for preparing cDNA libraries from scarce biological samples  

PubMed Central

The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids. PMID:25056322

Sterling, Catherine H.; Veksler-Lublinsky, Isana; Ambros, Victor

2015-01-01

215

Two-dimensional conformation-dependent electrophoresis (2D-CDE) to separate DNA fragments containing unmatched bulge from complex DNA samples  

PubMed Central

DNA fragments containing mispaired and modified bases, bulges, lesions and specific sequences have altered conformation. Methods for separating complex samples of DNA fragments based on conformation but independent of length have many applications, including (i) separation of mismatched or unmatched DNA fragments from those perfectly matched; (ii) simultaneous, diagnostic, mismatch scanning of multiple fragments; (iii) isolation of damaged DNA fragments from undamaged fragments; and (iv) estimation of reannealing efficiency of complex DNA samples. We developed a two-dimensional conformation-dependent electrophoresis (2D-CDE) method for separating DNA fragments based on length and conformation in the first dimension and only on length in the second dimension. Differences in migration velocity due to conformation were minimized during second dimension electrophoresis by introducing an intercalator. To test the method, we constructed 298 bp DNA fragments containing cytosine bulges ranging from 1 to 5 nt. Bulge-containing DNA fragments had reduced migration velocity in the first dimension due to altered conformation. After 2D-CDE, bulge-containing DNA fragments had migrated in front of an arc comprising heterogeneous fragments with regular conformation. This simple and robust method could be used in both analytical and preparative applications involving complex DNA samples. PMID:14762200

Gunnarsson, Gudmundur H.; Thormar, Hans G.; Gudmundsson, Bjarki; Akesson, Lina; Jonsson, Jon J.

2004-01-01

216

Robustness of genome-wide scanning using archived dried blood spot samples as a DNA source  

Microsoft Academic Search

Background  The search to identify disease-susceptible genes requires access to biological material from numerous well-characterized subjects.\\u000a Archived residual dried blood spot (DBS) samples, also known as Guthrie cards, from national newborn screening programs may\\u000a provide a DNA source for entire populations. Combined with clinical information from medical registries, DBS samples could\\u000a provide a rich source for productive research. However, the amounts

Mads V Hollegaard; Jakob Grove; Jonas Grauholm; Eskil Kreiner-Møller; Klaus Bønnelykke; Mette Nørgaard; Thomas L Benfield; Bent Nørgaard-Pedersen; Preben B Mortensen; Ole Mors; Henrik T Sørensen; Zitta B Harboe; Anders D Børglum; Ditte Demontis; Torben F Ørntoft; Hans Bisgaard; David M Hougaard

2011-01-01

217

A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions.  

PubMed Central

DNA from one biopsy sample of invasive cancer of the cervix contained sequences hybridizing with human papillomavirus (HPV) type 11 DNA only under nonstringent conditions. This DNA was molecularly cloned in lambda phage. Under stringent conditions of hybridization it cross-hybridized to a minor extent (less than 0.1%) with HPV types 10, 14, and 15 and showed no homology with DNA of other human HPV types. We therefore propose to designate it tentatively as HPV 16. HPV 16 DNA was used as a probe to test additional cancer biopsy samples from cervical, vulval, and penile cancer, as well as benign genital warts (condylomata acuminata) and cervical dysplasias for the presence of homologous sequences. In 61.1% (11/18) of cervical cancer samples from German patients sequences were found hybridizing with HPV 16 DNA under conditions of high stringency. In contrast, only 34.8% (8/23) of cancer biopsy samples from Kenya and Brazil revealed this DNA. Vulval and penile cancer biopsy samples hybridized to 28.6% (2/7) or 25% (1/4), respectively. Only 2 out of 33 condylomata acuminata contained HPV 16 DNA. Both positive tumors harbored in addition HPV 6 or HPV 11 DNA. The data thus indicate that HPV 16 DNA prevails in malignant tumors, rendering an accidental contamination with papillomavirus DNA from adjacent papillomas rather unlikely. The rare presence in benign genital papillomas in addition to common genital papillomaviruses suggests a dependence of HPV 16 replication on helper virus. Images PMID:6304740

Dürst, M; Gissmann, L; Ikenberg, H; zur Hausen, H

1983-01-01

218

Detection of Chlamydia psittaci DNA in avian clinical samples by polymerase chain reaction  

Microsoft Academic Search

A polymerase chain reaction (PCR) assay was developed to detect Chlamydia psittaci DNA in faeces and tissue samples from avian species. Primers were designed to amplify a 264 bp product derived from part of the 5? non-translated region and part of the coding region of the ompA gene which encodes the major outer membrane protein. Amplified sequences were confirmed by

R. Glyn Hewinson; Peter C. Griffiths; Bob J. Bevan; Sharon E. S. Kirwan; Mavis E. Field; Martin J. Woodward; Michael Dawson

1997-01-01

219

Importance Sampling of Word Patterns in DNA and Protein Sequences Hock Peng Chan  

E-print Network

when an asymptotic theory is absent or when the search sequence or the word pattern is too short. corresponding author. #12;1 Introduction Searching for matches to a word pattern in a stretch of biologicalImportance Sampling of Word Patterns in DNA and Protein Sequences Hock Peng Chan Department

Zhang, Nancy R.

220

Chronology-Sensitive Hierarchical Clustering of Pyrosequenced DNA Samples of E. coli: A Case Study  

E-print Network

Chronology-Sensitive Hierarchical Clustering of Pyrosequenced DNA Samples of E. coli: A Case Study; pyro- gram; I. INTRODUCTION Escherichia coli (E. coli) are commensal inhabitants of the human gut[1][2] and also thrive in the intestinal tracts of most other mammals and some birds[1][3]. E. coli is frequently

Dekhtyar, Alexander

221

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

Federal Register 2010, 2011, 2012, 2013, 2014

...Proposals involving whole-genome genotyping of DNA samples...proposals for whole-genome genotyping of more than...intends to provide whole genome-genotyping data from...protocol is closed and the project is transferred to the...submitted. Office of Human Research...

2010-06-07

222

Coordinate synthesis and turnover of heat shock proteins in Borrelia burgdorferi: degradation of DnaK during recovery from heat shock.  

PubMed Central

The synthesis and turnover of heat shock proteins (Hsps) by Borrelia burgdorferi, the Lyme disease spirochete, was investigated by radiolabeling of whole spirochetes and spheroplasts, comparison of one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and use of immunochemistry. The approximately 72-kDa DnaK homolog and three additional Hsps of 39, 27, and 21 kDa increased in amount by 3- to 15-fold between 2 and 6 h following temperature upshift from 28 to 39 degrees C. Temperature downshift experiments following the transfer of spirochetes from 40 to 28 degrees C showed that within 15 to 30 min, synthesis of most of the major Hsps returned to levels seen in spirochetes statically maintained at the lower temperature. Spheroplasts of B. burgdorferi produced by treatment with EDTA and lysozyme were radiolabeled, and specific Hsps were localized to either the cytoplasm or membrane fraction. Further analysis by two-dimensional electrophoresis demonstrated three constitutively expressed DnaK isoforms with pIs near 5.5. A pattern suggestive of DnaK degradation was observed following recovery from heat shock but not in spirochetes maintained entirely at a low temperature. Some of these putative degradation products were recognized by monoclonal antibodies directed against the B. burgdorferi DnaK protein. These data suggest that following a period of peak synthesis, DnaK is actively degraded as the spirochete reestablishes its metabolic thermometer. These findings provide a new interpretation of previous work suggesting that 10 to 15 B. burgdorferi polypeptides, including DnaK have a common epitope. PMID:8613385

Cluss, R G; Goel, A S; Rehm, H L; Schoenecker, J G; Boothby, J T

1996-01-01

223

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

224

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

225

Estimating Allele Frequency from Next-Generation Sequencing of Pooled Mitochondrial DNA Samples  

PubMed Central

Background: Both common and rare mitochondrial DNA (mtDNA) variants may contribute to genetic susceptibility to some complex human diseases. Understanding of the role of mtDNA variants will provide valuable insights into the etiology of these diseases. However, to date, there have not been any large-scale, genome-wide association studies of complete mtDNA variants and disease risk. One reason for this might be the substantial cost of sequencing the large number of samples required for genetic epidemiology studies. Next-generation sequencing of pooled mtDNA samples will dramatically reduce the cost of such studies and may represent an appealing approach for large-scale genetic epidemiology studies. However, the performance of the different designs of sequencing pooled mtDNA has not been evaluated. Methods: We examined the approach of sequencing pooled mtDNA of multiple individuals for estimating allele frequency using the Illumina genome analyzer (GA) II sequencing system. In this study the pool included mtDNA samples of 20 subjects that had been sequenced previously using Sanger sequencing. Each pool was replicated once to assess variation of the sequencing error between pools. To reduce such variation, barcoding was used for sequencing different pools in the same lane of the flow cell. To evaluate the effect of different pooling strategies pooling was done at both the pre- and post-PCR amplification step. Results: The sequencing error rate was close to that expected based on the Phred score. When only reads with Phred???20 were considered, the average error rate was about 0.3%. However, there was significant variation of the base-calling errors for different types of bases or at different loci. Using the results of the Sanger sequencing as the standard, the sensitivity of single nucleotide polymorphism detection with post-PCR pooling (about 99%) was higher than that of the pre-PCR pooling (about 82%), while the two approaches had similar specificity (about 99%). Among a total of 298 variants in the sample, the allele frequencies of 293 variants (98%) were correctly estimated with post-PCR pooling, the correlation between the estimated and the true allele frequencies being >0.99, while only 206 allele frequencies (69%) were correctly estimated in the pre-PCR pooling, the correlation being 0.89. Conclusion: Sequencing of mtDNA pooled after PCR amplification is a viable tool for screening mitochondrial variants potentially related to human diseases. PMID:22303347

Wang, Tao; Pradhan, Kith; Ye, Kenny; Wong, Lee-Jun; Rohan, Thomas E.

2011-01-01

226

Chromatographic separation of chlorophenoxy acid herbicides and their radiolytic degradation products in water samples.  

PubMed

HPLC procedure for simultaneous determination of chlorophenoxy acid herbicides and their radiolytic degradation products in waters is described with the use of octadecylsilica column and spectrophotometric detection at 280 nm. The satisfactory separation was achieved with a mobile phase of pH 2.5 consisting of 43.7 mM acetic acid with 40% (v/v) acetonitrile. Limit of detection values for herbicides and phenol derivatives were in the range of 19-41 microg/l and 10-60 microg/l, respectively. The developed method was applied for monitoring the effectiveness of radiolytic degradation of herbicides. Studies of products of gamma-radiolysis of 2,4-dichlorophenol have shown that the efficiency of this process is affected by the presence of naturally occurring scavengers of gamma-radiation such as carbonates or nitrates. PMID:15276742

Jankowska, A; Biesaga, M; Drzewicz, P; Trojanowicz, M; Pyrzy?ska, K

2004-01-01

227

Efficacy of DNA amplification in tissue biopsy samples to improve the detection of invasive fungal disease.  

PubMed

The performance of a pan-fungal PCR-based technique was evaluated to assess the aetiology of invasive fungal diseases (IFDs). A total of 89 formalin-fixed paraffin-embedded biopsy samples from 84 patients with proven IFD were studied. Culture of tissue was performed in 68 (81%) patients. The sensitivities of the PCR-based technique and microbiological culture of tissues were 89% and 56%, respectively (p <0.01). According to PCR results, Aspergillus species accounted for 67%, Candida species for 13%, zygomycetes for 11%, and rare and emerging fungi for 9%. Aspergillus species were significantly associated with lung samples (79.6%, p <0.01), Mucorales were associated with skin/subcutaneous samples, and Candida species were associated with gastrointestinal samples. Regarding biopsy samples with Aspergillus species, Aspergillus fumigatus DNA was detected in 43 of 50 (86%), and Aspergillus flavus in six of 50 (12%). PCR was positive in 24 of 30 (80%) cases with negative culture. In nine of the 84 patients, the PCR technique failed to amplify the DNA. Six also had negative cultures, and in the remaining three cases culture was positive (Rhizopus microsporus, Rhizopus arrhizus, and Sakseneae vasiformis), suggesting that the PCR technique was not as effective in amplifying the DNA of some species of Zygomycetes. In five cases, there was no correlation between culture results and those obtained with DNA amplification, indicating the possibility of a mixed infection or the presence of colonizer/contaminant microorganisms. In summary, PCR-based techniques for DNA amplification should be implemented in histopathology and microbiology departments, as they appear to be complementary to conventional methods for IFD detection. PMID:23464751

Buitrago, M J; Aguado, J M; Ballen, A; Bernal-Martinez, L; Prieto, M; Garcia-Reyne, A; Garcia-Rodriguez, J; Rodriguez-Tudela, J L; Cuenca-Estrella, M

2013-06-01

228

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

229

Isolation of Sperm Vesicles from Adult Male Mayflies and Other Insects to Prepare High Molecular Weight Genomic DNA Samples  

Microsoft Academic Search

We describe here a simple and efficient protocol for genomic DNA isolation from adult males of insects: e.g., Ephemeroptera,\\u000a Odonata, Orthoptera and Dictyoptera. To minimize contamination of external DNA source, the sperm vesicles were isolated from\\u000a male individuals from which high molecular weight genomic DNA was extracted. According to this protocol, the genomic DNA samples\\u000a obtained were high quality (intact),

Yasuhiro Takemon; Akiko Yamamoto; Masashi Nakashima; Kazumi Tanida; Mitsuo Kishi; Mikio Kato

2006-01-01

230

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

231

Methods for Integrated Air Sampling and DNA Analysis for Detection of Airborne Fungal Spores  

PubMed Central

Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting. PMID:11375150

Williams, Roger H.; Ward, Elaine; McCartney, H. Alastair

2001-01-01

232

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...

233

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

PubMed

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

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

2014-07-01

234

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

Microsoft Academic Search

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

Kangfu Yu; K. P. Pauls

1993-01-01

235

Apoptotic DNA Degradation into Oligonucleosomal Fragments, but Not Apoptotic Nuclear Morphology, Relies on a Cytosolic Pool of DFF40/CAD Endonuclease*  

PubMed Central

Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation. PMID:22253444

Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Gabernet, Gisela; García-Belinchón, Mercè; Sánchez-Osuna, María; Casanelles, Elisenda; Comella, Joan X.; Yuste, Victor J.

2012-01-01

236

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

PubMed

There is increasing interest in noninvasive DNA sampling techniques. In birds, there are several methods proposed for sampling DNA, and of these, the use of eggshell swabbing is potentially applicable to a wide range of species. We estimated the effectiveness of this method in the wild by sampling the eggs of 23 bird species. Sampling of eggs was performed twice per nest, soon after the clutch was laid and again at the end of egg incubation. We genotyped DNA samples using a set of five conserved microsatellite markers, which included a Z-linked locus and a sex-typing marker. We successfully collected avian DNA from the eggs of all species tested and from 88.48% of the samples. In most of the cases, the DNA concentration was low (ca. 10 ng/?L). The number of microsatellite loci amplified per sample (0-5) was used as a measure of the genotyping success of the sample. On average, we genotyped 3.01 ± 0.12 loci per sample (mean ± SE), and time of sampling did not seem to have an effect; however, genotyping success differed among species and was greater in those species that used feather material for lining their nest cups. We also checked for the occurrence of possible genotyping errors derived from using samples with very low DNA quantities (i.e. allelic dropout or false alleles) and for DNA contamination from individuals other than the mother, which appeared at a moderate rate (in 44% of the PCR replicates and in 17.36% of samples, respectively). Additionally, we investigated whether the DNA on eggshells corresponded to maternal DNA by comparing the genotypes obtained from the eggshells to those obtained from blood samples of all the nestlings for six nests of magpies. In five of the six magpie nests, we found evidence that the swab genotypes were a mixture of genotypes from both parents and this finding was independent of the time of incubation. Thus, our results broadly confirm that the swabbing of eggshells can be used as a noninvasive method for obtaining DNA and is applicable across a wide range of bird species. Nonetheless, genotyping errors should be properly estimated for each species by using a suite of highly polymorphic loci. These errors may be resolved by sampling only recently laid eggs (to avoid non-maternal DNA contamination) or by performing several PCR replicates per sample (to avoid allelic dropout and false alleles) and/or by increasing the amount of DNA used in the PCR through increasing the volume of the PCR or increasing the concentration of template DNA. PMID:21481206

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

2011-05-01

237

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

PubMed

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

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

2009-07-01

238

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

NASA Astrophysics Data System (ADS)

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

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

2010-04-01

239

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

240

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

241

Subtle changes to polymer structure and degradation mechanism enable highly effective nanoparticles for siRNA and DNA delivery to human brain cancer  

PubMed Central

Polymeric materials can be used to deliver nucleic acids such as DNA plasmids and siRNA, but often have low efficacy in human cells. To improve gene delivery, we synthesized an array of over 70 hydrolytically degradable and bioreducible poly(beta-amino ester)s and evaluated properties of over 200 nanoparticle formulations fabricated from these biomaterials. We determined the effect of different polymer structures on the delivery of nucleic acids of different structures and sizes, including siRNA, linear DNA, and circular DNAs (1.8–26 kb). Significantly, leading hydrolytically degradable polymeric nanoparticles delivered DNA to 90±2% of primary human glioblastoma cells with <10% nonspecific cytotoxicity, better than leading commercially available reagents (p<0.01). Bioreducible polymeric nanoparticles optimized for siRNA delivery caused up to 85±0.6% knockdown in these cells as well while maintaining high viability. From a single dose, knockdown was higher than for Lipofectamine™ 2000 (p<0.01) and persisted one month. Polymer molecular weight was a driving factor of transfection efficacy for some polymer structures (correlation of r2=0.63) but had no influence on transfection for other structures (r2=0.01). Polymers with a reducible cystamine functional group dramatically improved siRNA delivery by facilitating quick release while generally decreasing DNA delivery compared with non-reducible counterparts (p<0.01). Other material properties facilitated DNA delivery compared to siRNA delivery or increased delivery of both DNA and siRNA. PMID:23184674

Tzeng, Stephany Y.

2013-01-01

242

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

SciTech Connect

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

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

1993-07-01

243

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...

244

Reef-associated crustacean fauna: biodiversity estimates using semi-quantitative sampling and DNA barcoding  

Microsoft Academic Search

The cryptofauna associated with coral reefs accounts for a major part of the biodiversity in these ecosystems but has been\\u000a largely overlooked in biodiversity estimates because the organisms are hard to collect and identify. We combine a semi-quantitative\\u000a sampling design and a DNA barcoding approach to provide metrics for the diversity of reef-associated crustacean. Twenty-two\\u000a similar-sized dead heads of Pocillopora

L. Plaisance; N. Knowlton; G. Paulay; C. Meyer

2009-01-01

245

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

246

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

247

Establishment of a method of anonymization of DNA samples in genetic research  

Microsoft Academic Search

As the number of the genetic studies has rapidly increased in recent years, there has been growing concern that the privacy\\u000a of the participants in such studies can be invaded unless effective measures are adopted to protect confidentiality. It is\\u000a crucial for the scientific community to establish a method to anonymize DNA samples so that the public will trust genetic

Kazuo Hara; Kazuhiko Ohe; Takashi Kadowaki; Naoya Kato; Yasushi Imai; Katsushi Tokunaga; Ryozo Nagai; Masao Omata

2003-01-01

248

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

249

Variation of O 6 -methylguanine-DNA methyltransferase (MGMT) promoter methylation in serial samples in glioblastoma  

Microsoft Academic Search

Methylation of the promoter region of the O\\u000a \\u000a 6\\u000a \\u000a -methylguanine-DNA methyltransferase (MGMT) gene is known to be predictive of response to temozolomide treatment in patients with glioblastoma. Contrastingly, little\\u000a is known about variation in the methylation status of the MGMT promoter after treatment or across different regions of the same tumor. About 22 samples from 10 patients who had undergone

Jonathon F. Parkinson; Helen R. Wheeler; Adele Clarkson; Catriona A. McKenzie; Michael T. Biggs; Nicholas S. Little; Raymond J. Cook; Marinella Messina; Bruce G. Robinson; Kerrie L. McDonald

2008-01-01

250

A Column Experiment To Determine Black Shale Degradation And Colonization By Means of ?13C and 14C Analysis Of Phospholipid Fatty Acids And DNA Extraction  

NASA Astrophysics Data System (ADS)

We investigated the degradation of black shale organic matter by microbial communities. We inoculated two columns respectively, with the fungi Schizophyllum commune, the gram-positive bacterium Pseudomonas putida and the gram-negative bacteria Streptomyces griseus and Streptomyces chartreusis. These microorganisms are known to degrade a wide variety of organic macromolecules. Additionally, we had two sets of control columns. To one set the same nutrient solution was added as to the inoculated columns and to the other set only sterile deionised water was supplied. All columns contained 1.5 kg of freshly crushed not autoclaved black shale material with a particle size of 0.63-2 mm. The columns were incubated at 28° C and 60% humidity in the dark. The aim was to investigate, which microorganisms live on black shales and if these microorganisms are able to degrade ancient organic matter. We used compound specific stable isotope measurement techniques and compound specific 14C-dating methods. After 183 days PLFAs were extracted from the columns to investigate the microbial community, furthermore we extracted on one hand total-DNA of column material and on the other hand DNA from pure cultures isolates which grew on Kinks-agar B, Starch-casein-nitrate-agar (SCN) and on complete-yeast-medium-agar (CYM). According to the PLFA analysis bacteria dominated in the columns, whereas in pure cultures more fungi were isolated. A principal component analysis revealed differences between the columns in accordance with the inoculation, but it seems that the inoculated microorganisms were replaced by the natural population. For AMS measurements palmitic acid (C 16:0) was re-isolated from total-PLFA-extract with a preparative fraction collector (PFC). Preliminary results of the study revealed that microorganisms are able to degrade black shale material and that PLFA analysis are useful methods to be combined with analysis of stable isotope and 14C measurements to study microbial degradation processes.

Seifert, A.; Gleixner, G.

2008-12-01

251

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

252

Quantification of adducts formed in DNA treated with N-acetoxy-2-acetylaminofluorene or N-hydroxy-2-aminofluorene: comparison of trifluoroacetic acid and enzymatic degradation  

SciTech Connect

We have examined two methods of preparation of DNA adducts from phi X174 RF DNA modified by (/sup 3/H)N-acetoxy-2-acetylaminofluorene ((/sup 3/H)NA-AAF) or N-hydroxy-2-aminofluorene ((/sup 3/H)N-OH-AF). Hydrolysis by enzymes (DNase I, snake venom phosphodiesterase and alkaline or acid phosphatase) and subsequent reverse phase h.p.l.c. of phi X174 RF DNA treated with (/sup 3/H)NA-AAF yielded 73% N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), 7% 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene (dG-N2-AAF), and a peak of unidentified radioactivity (13%). When (/sup 3/H)N-OH-AF modified phi X174 DNA was analyzed, both N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and a large percentage of the imidazole ring-opened derivative and unidentified products were found. In contrast, when anhydrous trifluoroacetic acid (TFA) was used to degrade these DNAs, we found for the (/sup 3/H)NA-AAF modified DNA 86% N-(guanin-8-yl)-2-acetylaminofluorene (G-C8-AAF) and 6% 3-(guanin-N2-yl)-2-acetylaminofluorene (G-N2-AAF), while for (/sup 3/H)N-OH-AF modified DNA only the N-(guanin-8-yl)-2-aminofluorene (G-C8-AF) was found. When DNA was prepared from human fibroblasts treated with (/sup 3/H)NA-AAF, only the G-C8-AF product was obtained. Thus, anhydrous TFA solvolysis followed by reverse phase h.p.l.c. is a rapid and convenient method to obtain quantitative yields of DNA adducts formed with acetylaminofluorene and related compounds: quantification by this method prevents loss of G-N2-AAF adducts, the conversion of AAF adducts to AF adducts, and the production of ring opened products in guanine residue.

Tang, M.; Lieberman, M.W.

1983-01-01

253

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

PubMed Central

Background Deciphering the behavioral repertoire of great apes is a challenge for several reasons. First, due to their elusive behavior in dense forest environments, great ape populations are often difficult to observe. Second, members of the genus Pan are known to display a great variety in their behavioral repertoire; thus, observations from one population are not necessarily representative for other populations. For example, bonobos (Pan paniscus) are generally believed to consume almost no vertebrate prey. However, recent observations show that at least some bonobo populations may consume vertebrate prey more commonly than previously believed. We investigated the extent of their meat consumption using PCR amplification of vertebrate mitochondrial DNA (mtDNA) segments from DNA extracted from bonobo feces. As a control we also attempted PCR amplifications from gorilla feces, a species assumed to be strictly herbivorous. Principal Findings We found evidence for consumption of a variety of mammalian species in about 16% of the samples investigated. Moreover, 40% of the positive DNA amplifications originated from arboreal monkeys. However, we also found duiker and monkey mtDNA in the gorilla feces, albeit in somewhat lower percentages. Notably, the DNA sequences isolated from the two ape species fit best to the species living in the respective regions. This result suggests that the sequences are of regional origin and do not represent laboratory contaminants. Conclusions Our results allow at least three possible and mutually not exclusive conclusions. First, all results may represent contamination of the feces by vertebrate DNA from the local environment. Thus, studies investigating a species' diet from feces DNA may be unreliable due to the low copy number of DNA originating from diet items. Second, there is some inherent difference between the bonobo and gorilla feces, with only the later ones being contaminated. Third, similar to bonobos, for which the consumption of monkeys has only recently been documented, the gorilla population investigated (for which very little observational data are as yet available) may occasionally consume small vertebrates. Although the last explanation is speculative, it should not be discarded a-priori given that observational studies continue to unravel new behaviors in great ape species. PMID:20195539

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

2010-01-01

254

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

PubMed Central

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

Kuske, Cheryl R.; Banton, Kaysie L.; Adorada, Dante L.; Stark, Peter C.; Hill, Karen K.; Jackson, Paul J.

1998-01-01

255

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

256

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

NASA Astrophysics Data System (ADS)

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.

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

2011-05-01

257

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

258

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

259

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

260

The human interferon- and estrogen-regulated ISG20/HEM45 gene product degrades single-stranded RNA and DNA in vitro.  

PubMed

The human ISG20/HEM45 gene was identified independently on the basis of its increased level of expression in response to either interferon or estrogen hormone. Notably, the encoded protein is homologous with members of the 3' to 5' exonuclease superfamily that includes RNases T and D, and the proofreading domain of Escherichia coli DNA polymerase I. We provide here direct biochemical evidence that Isg20 acts as a 3' to 5' exonuclease in vitro. This protein displays a pH optimum of approximately 7.0, prefers Mn2+ as a metal cofactor, and degrades RNA at a rate that is approximately 35-fold higher than its rate for single-stranded DNA. Along with RNase L, Isg20 is the second known RNase regulated by interferon. Previous data showed that Isg20 is located in promyelocytic leukemia (PML) nuclear bodies, known sites of hormone-dependent RNA polymerase II transcription and oncogenic DNA viral transcription and replication. The combined data suggest a potential role for Isg20 in degrading viral RNAs as part of the interferon-regulated antiviral response and/or cellular mRNAs as a regulatory component of interferon and estrogen signaling. PMID:11401564

Nguyen, L H; Espert, L; Mechti, N; Wilson, D M

2001-06-19

261

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

262

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

PubMed Central

The availability of a useful tool for simple and timely detection of the most important virulent varieties of Escherichia coli is indispensable. To this end, bacterial DNA pools which had previously been categorized were obtained from isolated colonies as well as selected in terms of utilized phenotype; the pools were assessed by two PCR Multiplex for the detection of virulent E. coli eaeA, bfpA, stx1, stx2, ipaH, ST, LT, and aatA genes, with the 16S gene used as DNA control. The system was validated with 66 fecal samples and 44 wastewater samples. At least one positive isolate was detected by a virulent gene among the 20 that were screened. The analysis of fecal samples from children younger than 6 years of age detected frequencies of 25% LT positive strains, 8.3% eae, 8.3% bfpA, 16.7% ipaH, as well as 12.5 % aatA and ST. On the other hand, wastewater samples revealed frequencies of 25.7% eaeA positive, 30.3% stx1, 15.1% LT and 19.7% aatA. This study is an initial step toward carrying out epidemiological field research that will reveal the presence of these bacterial varieties. PMID:24031959

Luz María Chacón, J; Lizeth Taylor, C; Carmen Valiente, A; Irene Alvarado, P; Ximena Cortés, B

2012-01-01

263

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

264

Microsatellite-Based Parentage Analysis of Aedes aegypti (Diptera: Culicidae) Using Nonlethal DNA Sampling  

PubMed Central

To track Aedes aegypti (L.) egg-laying behavior in the field in Iquitos, Peru, we developed methods for 1) sampling DNA from live mosquitoes and 2) high through-put parentage analysis using microsatellite markers. We were able to amplify DNA extracted from a single hind leg, but not from the pupal exuvia. Removal of a leg from teneral females caused no significant changes in female behavioral or life history traits (e.g., longevity, blood feeding frequency, fecundity, egg hatch rate, gonotrophic cycle length, or oviposition behavior). Using a panel of nine microsatellite markers and an exclusion-based software program, we matched offspring to parental pairs in 10 Ae. aegypti test families in which parents originated from natural development sites in Iquitos. By mating known individuals in the laboratory, retaining the male, sampling the female’s DNA before release, and collecting offspring in the field, the technique we developed can be used to genotype large numbers of Ae. aegypti, reconstruct family relationships, and track the egg-laying behavior of individual Ae. aegypti in nature. PMID:22308775

WONG, JACKLYN; CHU, YUI YIN; STODDARD, STEVEN T.; LEE, YOOSOOK; MORRISON, AMY C.; SCOTT, THOMAS W.

2012-01-01

265

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

266

Comparative analysis of three methods for HPV DNA detection in cervical samples.  

PubMed

High risk HPV infection is considered to play a central role in cervical carcinogenesis. HPV DNA testing has shown to be a very useful tool for screening and following cervical infections. The aim of this study was to compare three methods for HPV DNA detection, along with cytology and colposcopy analysis. Cervical samples were collected from 100 sexually active women in Mérida, western Venezuela. HPV infection was screened using Hybrid-Capture 2 (HC2), L1-Nested-PCR and E6/E7-PCR assays. 40% of the samples (40/100) were HPV positive by at least one of the DNA detection methods. HC2 detected HPV in 12% specimens. L1- and E6/E7-PCRs showed 50% sensitivity and 77% specificity.The agreement rate between HC2 and both PCR assays was 65%. Kappa value showed moderate concordance between HC2 and both PCR methods (kappa=0.55; CI 95%). Also moderate concordance was seen when L1- and E6/E7-PCRs were compared (kappa=0.48; CI 95%). There was a significant association between the Schiller test and E6/E7-PCR (p=0.006) for HPV infection. An acceptable agreement between all three assays for HPV detection was observed. Nevertheless, different PCR formats need to be further analyzed in order to make the right choice of method for HPV testing. PMID:22523844

Michelli, Elvia; Téllez, Luis; Mendoza, José-Andrés; Jürgensen, Claudia; Muñoz, Maritza; Pérez, Saberio; Mosqueda, Noraida; Hernández, Erick; Noguera, María-Eugenia; Callejas, Diana; Correnti, María; Cavazza, María-Eugenia; Vielma, Silvana

2011-12-01

267

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

E-print Network

using selective precipitation by compaction agents. Añez-Lingerfelt M, Fox GE, Willson RC. Department-effective method of eliminating contaminating DNA in RNA samples using selective precipitation by compaction agents precipitation of salmon sperm DNA. Effectiveness and selectivity were then investigated using differences in RT

Fox, George

268

Quantification of heteroplasmic mitochondrial DNA mutations for DNA samples in the low picogram range by nested real-time ARMS-qPCR.  

PubMed

In many mitochondrial diseases, different clinical manifestations are related to tissue-specific distribution of mutated mitochondrial DNA (mtDNA). In this study, we describe an assay for the determination of mutated mtDNA copy number in small clinical samples, using standard polymerase chain reaction (PCR) followed by SYBR Green real-time allelic-specific PCR [amplification refractory mutation system-quantitative PCR (ARMS-qPCR)]. To assess the degree of heteroplasmy in a patient harboring 2 cosegregating mtDNA mutations (4415A>G and 9922A>C) starting from picogram amounts of DNA, we first amplified the mutated target sequence by standard PCR, and then analyzed it by real-time ARMS-qPCR. To validate this method, we analyzed by real-time ARMS-qPCR the PCR amplification products derived from different mixtures containing known proportions of mutant and wild-type cloned mtDNA fragments. The correlation coefficient of 0.994 between expected and observed values for the percentage of mutant A4415G confirms that the relative proportion of mutated and wild-type mtDNA was maintained after the first PCR amplification. This method allows the precise quantification of heteroplasmic mutations in DNA samples extracted from hairs, urine, small stomach biopsies, and, more importantly, single-muscle fiber, with a limit of detection close to 0.5%. This nested real-time ARMS-PCR represents a rapid, efficient, and less expensive method for the detection and quantification of heteroplasmic mutant mtDNA, even in very small clinical samples. PMID:21532488

Biffi, Stefania; Bortot, Barbara; Carrozzi, Marco; Severini, Giovanni Maria

2011-06-01

269

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

PubMed Central

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

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

2011-01-01

270

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

PubMed Central

A group of antibiotic resistance genes (ARGs) (blaTEM, blaCTX-M-1, mecA, armA, qnrA, and qnrS) were analyzed by real-time quantitative PCR (qPCR) in bacteriophage DNA isolated from feces from 80 healthy humans. Seventy-seven percent of the samples were positive in phage DNA for one or more ARGs. blaTEM, qnrA, and, blaCTX-M-1 were the most abundant, and armA, qnrS, and mecA were less prevalent. Free bacteriophages carrying ARGs may contribute to the mobilization of ARGs in intra- and extraintestinal environments. PMID:24165177

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

2014-01-01

271

Amplifiability of mitochondrial, microsatellite and amelogenin DNA loci from fecal samples of red brocket deer Mazama americana (Cetartiodactyla, Cervidae).  

PubMed

We tried to amplify mitochondrial, microsatellite and amelogenin loci in DNA from fecal samples of a wild Mazama americana population. Fifty-two deer fecal samples were collected from a 600-ha seasonal semideciduous forest fragment in a subtropical region of Brazil (21°20'S, 47°17'W), with the help of a detection dog; then, stored in ethanol and georeferenced. Among these samples 16 were classified as "fresh" and 36 as "non-fresh". DNA was extracted using the QIAamp(®) DNA Stool Mini Kit. Mitochondrial loci were amplified in 49 of the 52 samples. Five microsatellite loci were amplified by PCR; success in amplification varied according to locus size and sample age. Successful amplifications were achieved in 10/16 of the fresh and in 13/36 of the non-fresh samples; a negative correlation (R = -0.82) was found between successful amplification and locus size. Amplification of the amelogenin locus was successful in 22 of the 52 samples. The difficulty of amplifying nuclear loci in DNA samples extracted from feces collected in the field was evident. Some methodological improvements, including collecting fresh samples, selecting primers for shorter loci and quantifying the extracted DNA by real-time PCR, are suggested to increase amplification success in future studies. PMID:23359023

Oliveira, M L; Duarte, J M B

2013-01-01

272

Comparison of four DNA extraction methods for forensic application  

Microsoft Academic Search

Challenging biological samples found in crime scenes are often brought to our lab. Several factors, such as degradation and the presence of inhibitors, can difficult the analysis of these samples. Chelating resin, silica membranes, silica-coated magnetic beads and paramagnetic resin were DNA extraction techniques used in this study. Our aim was to find out the DNA extraction method more suitable

V. Bogas; F. Balsa; M. Carvalho; M. J. Anjos; M. F. Pinheiro; F. Corte-Real

273

Evaluation of a semi-automated, magnetic bead-based DNA extraction method for genetic fingerprinting of forensic casework samples  

Microsoft Academic Search

In order to cope with the demanding workload for DNA profiling of forensic casework samples a concept for a semi-automated processing system was developed at the Landeskriminalamt (Office of Criminal Investigation) Baden-Württemberg, Germany [K. Vollack, et al., Implementation of a semi-automated processing system for DNA profiling of forensic casework samples, this issue]. The applied magnetic bead extraction method is based

Barbara Haak; Andrea Porsche; Kai Vollack; Peter Zimmermann; Werner Pflug

2008-01-01

274

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

PubMed Central

Background DNA barcoding is expected to be an effective identification tool for organisms with heteromorphic generations such as pteridophytes, which possess a morphologically simple gametophyte generation. Although a reference data set including complete coverage of the target local flora/fauna is necessary for accurate identification, DNA barcode studies including such rich taxonomic sampling on a countrywide scale are lacking. Methodology/Principal Findings The Japanese pteridophyte flora (733 taxa including subspecies and varieties) was used to test the utility of two plastid DNA barcode regions (rbcL and trnH-psbA) with the intention of developing an identification system for native gametophytes. DNA sequences were obtained from each of 689 (94.0%) taxa for rbcL and 617 (84.2%) taxa for trnH-psbA. Mean interspecific divergence values across all taxon pairs (K2P genetic distances) did not reveal a significant difference in rate between trnH-psbA and rbcL, but mean K2P distances of each genus showed significant heterogeneity according to systematic position. The minimum fail rate of taxon discrimination in an identification test using BLAST (12.52%) was obtained when rbcL and trnH-psbA were combined, and became lower in datasets excluding infraspecific taxa or apogamous taxa, or including sexual diploids only. Conclusions/Significance This study demonstrates the overall effectiveness of DNA barcodes for species identification in the Japanese pteridophyte flora. Although this flora is characterized by a high occurrence of apogamous taxa that pose a serious challenge to identification using DNA barcodes, such taxa are limited to a small number of genera, and only minimally detract from the overall success rate. In the case that a query sequence is matched to a known apogamous genus, routine species identification may not be possible. Otherwise, DNA barcoding is a practical tool for identification of most Japanese pteridophytes, and is especially anticipated to be helpful for identification of non-hybridizing gametophytes. PMID:21170336

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

2010-01-01

275

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

276

C/EBP? regulates CRL4(Cdt2)-mediated degradation of p21 in response to UVB-induced DNA damage to control the G1/S checkpoint.  

PubMed

The bZIP transcription factor, C/EBP? is highly inducible by UVB and other DNA damaging agents in keratinocytes. C/EBP?-deficient keratinocytes fail to undergo cell cycle arrest in G1 in response to UVB-induced DNA damage and mice lacking epidermal C/EBP? are highly susceptible to UVB-induced skin cancer. The mechanism through which C/EBP? regulates the cell cycle checkpoint in response to DNA damage is unknown. Here we report untreated C/EBP?-deficient keratinocytes have normal levels of the cyclin-dependent kinase inhibitor, p21, however, UVB-treated C/EBP?-deficient keratinocytes fail to up-regulate nuclear p21 protein levels despite normal up-regulation of Cdkn1a mRNA levels. UVB-treated C/EBP?-deficient keratinocytes displayed a 4-fold decrease in nuclear p21 protein half-life due to the increased proteasomal degradation of p21 via the E3 ubiquitin ligase CRL4(Cdt2). Cdt2 is the substrate recognition subunit of CRL4(Cdt2) and Cdt2 mRNA and protein levels were up-regulated in UVB-treated C/EBP?-deficient keratinocytes. Knockdown of Cdt2 restored p21 protein levels in UVB-treated C/EBP?-deficient keratinocytes. Lastly, the failure to accumulate p21 in response to UVB in C/EBP?-deficient keratinocytes resulted in decreased p21 interactions with critical cell cycle regulatory proteins, increased CDK2 activity, and inappropriate entry into S-phase. These findings reveal C/EBP? regulates G1/S cell cycle arrest in response to DNA damage via the control of CRL4(Cdt2) mediated degradation of p21. PMID:25483090

Hall, Jonathan R; Bereman, Michael S; Nepomuceno, Angelito I; Thompson, Elizabeth A; Muddiman, David C; Smart, Robert C

2014-01-01

277

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

278

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

PubMed Central

Four DNA probes were derived that hybridized specifically to DNA from Lactobacillus acidophilus O. The probes were constructed by randomly cloning lactobacillus DNA in plasmid vector pBR322. Two of the probes (pSR1 and pSR2) were composed of vector and plasmid DNA inserts (3.6 and 1.6 kb, respectively); the others (pSR3 and pSR4) were composed of vector and chromosomally derived inserts (6.9 and 1.4 kb, respectively). The probes were used to enumerate, by colony hybridization, strain O in digestive tract samples collected from piglets inoculated 24 hours previously with a culture of the strain. The probes did not hybridize to DNA from lactobacilli inhabiting the digestive tract of uninoculated piglets. Strain O made up about 10% of the total lactobacillus population of the pars esophagea and about 20% of the population in other digestive tract samples. Images PMID:8285690

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

1993-01-01

279

Comparative study of seven commercial kits for human DNA extraction from urine samples suitable for DNA biomarker-based public health studies.  

PubMed

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

2014-12-01

280

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

281

Expanding Character Sampling for Ciliate Phylogenetic Inference Using Mitochondrial SSU-rDNA as a Molecular Marker  

PubMed Central

Molecular systematics of ciliates, particularly at deep nodes, has largely focused on increasing taxon sampling using the nuclear small subunit rDNA (nSSU-rDNA) locus. These previous analyses have generally been congruent with morphologically-based classifications, although there is extensive non-monophyly at many levels. However, caution is needed in interpreting these results as nSSU-rDNA is just a single molecular marker. Here the mitochondrial small subunit rDNA (mtSSU-rDNA) is evaluated for deep ciliate nodes using the Colpodea as an example. Overall, well-supported nodes in the mtSSU-rDNA and concatenated topologies are well supported in the nSSU-rDNA topology; e.g., the non-monophyly of the Cyrtolophosidida. The two moderately-to well-supported incongruences between the loci are the placement of the Sorogenida and Colpoda aspera. Our analyses of mtSSU-rDNA support the conclusion, originally derived from nSSU-rDNA, that the morphological characters used in taxonomic circumscriptions of the Colpodea represent a mixture of ancestral and derived states. This demonstration of the efficacy of the mtSSU-rDNA will enable phylogenetic reconstructions of deep nodes in the ciliate tree of life to move from a single-locus to a multi-locus approach. PMID:20708960

Dunthorn, Micah; Foissner, Wilhelm; Katz, Laura A.

2012-01-01

282

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

283

High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique  

SciTech Connect

Sample preparation has been one of the major bottlenecks for many high throughput analyses. The purpose of this research was to develop new sample preparation and integration approach for DNA sequencing, PCR based DNA analysis and combinatorial screening of homogeneous catalysis based on multiplexed capillary electrophoresis with laser induced fluorescence or imaging UV absorption detection. The author first introduced a method to integrate the front-end tasks to DNA capillary-array sequencers. protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. After the colony was picked, lysis was accomplished in situ in the plastic sample tube using either a thermocycler or heating block. Upon heating, the plasmids were released while chromsomal DNA and membrane proteins were denatured and precipitated to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed on-line DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation. For PCR based DNA analysis, they demonstrated that capillary electrophoresis with UV detection can be used for DNA analysis starting from clinical sample without purification. After PCR reaction using cheek cell, blood or HIV-1 gag DNA, the reaction mixtures was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis. The use of cheaper detection, and the elimination of purification of DNA sample before or after PCR reaction, will make this approach an attractive alternative to current methods for genetic analysis and disease diagnosis.

Yonghua Zhang

2002-05-27

284

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

285

Feline mitochondrial DNA sampling for forensic analysis: When enough is enough!  

PubMed

Pet hair has a demonstrated value in resolving legal issues. Cat hair is chronically shed and it is difficult to leave a home with cats without some level of secondary transfer. The power of cat hair as an evidentiary resource may be underused because representative genetic databases are not available for exclusionary purposes. Mitochondrial control region databases are highly valuable for hair analyses and have been developed for the cat. In a representative worldwide data set, 83% of domestic cat mitotypes belong to one of twelve major types. Of the remaining 17%, 7.5% are unique within the published 1394 sample database. The current research evaluates the sample size necessary to establish a representative population for forensic comparison of the mitochondrial control region for the domestic cat. For most worldwide populations, randomly sampling 50 unrelated local individuals will achieve saturation at 95%. The 99% saturation is achieved by randomly sampling 60-170 cats, depending on the numbers of mitotypes available in the population at large. Likely due to the recent domestication of the cat and minimal localized population substructure, fewer cats are needed to meet mitochondria DNA control region database practical saturation than for humans or dogs. Coupled with the available worldwide feline control region database of nearly 1400 cats, minimal local sampling will be required to establish an appropriate comparative representative database and achieve significant exclusionary power. PMID:25531059

Grahn, Robert A; Alhaddad, Hasan; Alves, Paulo C; Randi, Ettore; Waly, Nashwa E; Lyons, Leslie A

2015-05-01

286

Mitochondrial DNA control region variation in an autochthonous Basque population sample from the Basque Country.  

PubMed

Mitochondrial control region (16024-576) sequences were generated from 106 samples from autochthonous Basques from the Autonomous Community of the Basque Country. It is especially important to generate mtDNA databases from isolated populations in order to maximize the power of discrimination of this molecular marker. It also represents a useful approach to carry out a more accurate haplogroup classification. This is the first database report of complete control region sequences in an autochthonous Basque population sample. Strict selection criteria of autochthonous individuals, automation of laboratory processing and independent reviews of the raw electropherograms ensure the high quality of these sequences and their utility as reference population data of the autochthonous Basque population. PMID:22277258

Cardoso, Sergio; Villanueva-Millán, María Jesús; Valverde, Laura; Odriozola, Adrian; Aznar, Jose M; Piñeiro-Hermida, Sergio; de Pancorbo, Marian M

2012-07-01

287

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

288

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

289

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.

Jennifer Doherty

290

Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation.  

PubMed Central

We show that upon induction of Agrobacterium tumefaciens, free linear double-stranded T-DNA molecules as well as the previously described T-strands are generated from the Ti plasmid. A majority of these molecules are bound to a protein. We show that this protein is the product of the virulence gene virD2. This protein was found to be attached to the 5' terminus of processed T-DNA at the right border and to the rest of the Ti plasmid at the left border. The protein remnant after Pronase digestion rendered the right end of the double-stranded T-DNA resistant to 5'----3' exonucleolytic attack in vitro. The protein-DNA association was resistant to SDS, mercaptoethanol, mild alkali, piperidine, and hydroxylamine, indicating that it involves a covalent linkage. The possible involvement of this T-DNA-protein complex in replication, transduction to the plant, nuclear targeting, and integration into the plant nuclear DNA is discussed. Images PMID:2556703

Dürrenberger, F; Crameri, A; Hohn, B; Koukolíková-Nicola, Z

1989-01-01

291

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

292

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

293

A DNA based method to detect the grapevine root-rotting fungus Roesleria subterranea in soil and root samples  

PubMed Central

Summary Roesleria subterranea causes root rot in grapevine and fruit trees. The fungus has long been underestimated as a weak parasite, but during the last years it has been reported to cause severe damages in German vineyards. Direct, observation-based detection of the parasite is time consuming and destructive, as large parts of the rootstocks have to be uprooted and screened for the tiny, stipitate, hypogeous ascomata of R. subterranea. To facilitate rapid detection in vineyards, protocols to extract DNA from soil samples and grapevine roots, and R.-subterranea-specific PCR primers were designed. Twelve DNA–extraction protocols for soil samples were tested in small-scale experiments, and selected parameters were optimised. A protocol based on ball-mill homogenization, DNA extraction with SDS, skim milk, chloroform, and isopropanol, and subsequent purification of the raw extracts with PVPP-spin-columns was most effective. This DNA extraction protocol was found to be suitable for a wide range of soil-types including clay, loam and humic-rich soils. For DNA extraction from grapevine roots a CTAB-based protocol was more reliable for various grapevine rootstock varieties. Roesleria-subterranea-specific primers for the ITS1–5.8S–ITS2 rDNA-region were developed and tested for their specificity to DNA extracts from eleven R. subterranea strains isolated from grapevine and fruit trees. No cross reactions were detected with DNA extracts from 44 different species of fungi isolated from vineyard soils. The sensitivity of the species-specific primers in combination with the DNA extraction method for soil was high: as little as 100 fg ?l?1 R.-subterranea-DNA was sufficient for a detection in soil samples and plant material. Given that specific primers are available, the presented method will also allow quick and large-scale testing for other root pathogens. PMID:21442023

Neuhauser, Sigrid; Huber, Lars; Kirchmair, Martin

2011-01-01

294

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

295

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

Microsoft Academic Search

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

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

2008-01-01

296

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

Microsoft Academic Search

BACKGROUND: Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in

Katarina Cankar; Dejan Štebih; Tanja Dreo; Jana Žel; Kristina Gruden

2006-01-01

297

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

298

Reef-associated crustacean fauna: biodiversity estimates using semi-quantitative sampling and DNA barcoding  

NASA Astrophysics Data System (ADS)

The cryptofauna associated with coral reefs accounts for a major part of the biodiversity in these ecosystems but has been largely overlooked in biodiversity estimates because the organisms are hard to collect and identify. We combine a semi-quantitative sampling design and a DNA barcoding approach to provide metrics for the diversity of reef-associated crustacean. Twenty-two similar-sized dead heads of Pocillopora were sampled at 10 m depth from five central Pacific Ocean localities (four atolls in the Northern Line Islands and in Moorea, French Polynesia). All crustaceans were removed, and partial cytochrome oxidase subunit I was sequenced from 403 individuals, yielding 135 distinct taxa using a species-level criterion of 5% similarity. Most crustacean species were rare; 44% of the OTUs were represented by a single individual, and an additional 33% were represented by several specimens found only in one of the five localities. The Northern Line Islands and Moorea shared only 11 OTUs. Total numbers estimated by species richness statistics (Chao1 and ACE) suggest at least 90 species of crustaceans in Moorea and 150 in the Northern Line Islands for this habitat type. However, rarefaction curves for each region failed to approach an asymptote, and Chao1 and ACE estimators did not stabilize after sampling eight heads in Moorea, so even these diversity figures are underestimates. Nevertheless, even this modest sampling effort from a very limited habitat resulted in surprisingly high species numbers.

Plaisance, L.; Knowlton, N.; Paulay, G.; Meyer, C.

2009-12-01

299

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

300

A novel method of selective removal of human DNA improves PCR sensitivity for detection of Salmonella Typhi in blood samples  

PubMed Central

Background Enteric fever is a major public health problem, causing an estimated 21million new cases and 216,000 or more deaths every year. Current diagnosis of the disease is inadequate. Blood culture only identifies 45 to 70% of the cases and is time-consuming. Serological tests have very low sensitivity and specificity. Clinical samples obtained for diagnosis of enteric fever in the field generally have <1 organism/ml of blood, so that even PCR-based methods, widely used for detection of other infectious diseases, are not a straightforward option in typhoid diagnosis. We developed a novel method to enrich target bacterial DNA by selective removal of human DNA from blood samples, enhancing the sensitivity of PCR tests. This method offers the possibility of improving PCR assays directly using clinical specimens for diagnosis of this globally important infectious disease. Methods Blood samples were mixed with ox bile for selective lysis of human blood cells and the released human DNA was then digested with addition of bile resistant micrococcal nuclease. The intact Salmonella Typhi bacteria were collected from the specimen by centrifugation and the DNA extracted with QIAamp DNA mini kit. The presence of Salmonella Typhi bacteria in blood samples was detected by PCR with the fliC-d gene of Salmonella Typhi as the target. Results Micrococcal nuclease retained activity against human blood DNA in the presence of up to 9% ox bile. Background human DNA was dramatically removed from blood samples through the use of ox bile lysis and micrococcal nuclease for removal of mammalian DNA. Consequently target Salmonella Typhi DNA was enriched in DNA preparations and the PCR sensitivity for detection of Salmonella Typhi in spiked blood samples was enhanced by 1,000 fold. Conclusions Use of a combination of selective ox-bile blood cell lysis and removal of human DNA with micrococcal nuclease significantly improves PCR sensitivity and offers a better option for improved typhoid PCR assays directly using clinical specimens in diagnosis of this globally important infection disease which we believe could be of importance in improving clinical care and providing effective evaluation of novel vaccines. PMID:22839649

2012-01-01

301

Development of tetranucleotide microsatellite loci and a non-invasive DNA sampling method for Texas horned lizards ( Phrynosoma cornutum )  

Microsoft Academic Search

We developed a non-invasive DNA sampling method and 15 tetranucleotide microsatellite markers for Texas horned lizards (Phrynosoma cornutum). Swabbing the cloaca with a small cotton swab and preserving the cells in lysis buffer was an effective method to obtain\\u000a tissue for DNA extraction. Loci were highly polymorphic with 8–25 alleles and observed heterozygosity was high (0.71–0.96).\\u000a Some of these loci

Dean A. Williams; Cory Leach; Amanda M. Hale; Kristopher B. Karsten; Emmanuela Mujica; Diane Barber; Lee Ann Linam; Nathan Rains

302

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

303

Association screen for atopic dermatitis candidate gene regions using microsatellite markers in pooled DNA samples.  

PubMed

Atopic dermatitis (AD) is a chronic inflammatory skin disease affecting up to 16% of children in developed countries. A complex genetic background for AD has been suggested, with genetic as well as environmental factors influencing disease susceptibility. Among other factors, dysregulation in both the innate and the adaptive immune system has been proposed to play a role in AD pathophysiology. We present here an extended association screen for AD using microsatellite markers in 154 genes related to innate and adaptive immunity in pooled DNA samples from 150 German children with AD and 100 controls. After Bonferroni correction, no marker revealed a significant association with AD. Yet, markers representing the nuclear factor kappa B (NFKB)1 and chemokine receptor (CCR)4 genes showed differences in allelic distributions between cases and controls for both pooled DNA analysis and individual genotyping and were thus further investigated. Evaluation of additional single nucleotide polymorphisms (SNP) in the NFKB1 and CCR4 genes revealed no association of individual SNPs with AD. In contrast, haplotype analyses showed a significantly different haplotype distribution between patients and controls for CCR4 (P < 0.001). Furthermore, when SNP-SNP interaction effects were analysed for these two genes, we found significant evidence for epistatic interactions between SNPs within each of the two genes but no evidence for a gene-gene interaction, suggesting that variation in or near both the CCR4 and the NFKB1 genes might individually contribute to AD pathogenesis. PMID:17117949

Hoffjan, S; Parwez, Q; Petrasch-Parwez, E; Falkenstein, D; Nothnagel, M; Epplen, J T

2006-12-01

304

Partition Enrichment of Nucleotide Sequences (PINS) - A Generally Applicable, Sequence Based Method for Enrichment of Complex DNA Samples  

PubMed Central

The dwindling cost of DNA sequencing is driving transformative changes in various biological disciplines including medicine, thus resulting in an increased need for routine sequencing. Preparation of samples suitable for sequencing is the starting point of any practical application, but enrichment of the target sequence over background DNA is often laborious and of limited sensitivity thereby limiting the usefulness of sequencing. The present paper describes a new method, Probability directed Isolation of Nucleic acid Sequences (PINS), for enrichment of DNA, enabling the sequencing of a large DNA region surrounding a small known sequence. A 275,000 fold enrichment of a target DNA sample containing integrated human papilloma virus is demonstrated. Specifically, a sample containing 0.0028 copies of target sequence per ng of total DNA was enriched to 786 copies per ng. The starting concentration of 0.0028 target copies per ng corresponds to one copy of target in a background of 100,000 complete human genomes. The enriched sample was subsequently amplified using rapid genome walking and the resulting DNA sequence revealed not only the sequence of a the truncated virus, but also 1026 base pairs 5? and 50 base pairs 3? to the integration site in chromosome 8. The demonstrated enrichment method is extremely sensitive and selective and requires only minimal knowledge of the sequence to be enriched and will therefore enable sequencing where the target concentration relative to background is too low to allow the use of other sample preparation methods or where significant parts of the target sequence is unknown. PMID:25203653

Kvist, Thomas; Sondt-Marcussen, Line; Mikkelsen, Marie Just

2014-01-01

305

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

306

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

E-print Network

the feasibility of using saliva-extracted DNA in comparison to blood-derived DNA, across two genotyping platforms: Applied Biosystems TaqmanTM and Illumina BeadchipTM genome-wide arrays. Method Patients were recruited from the Pharmacogenetics of Breast Cancer...

Abraham, Jean E; Maranian, Mel J; Spiteri, Inmaculada; Russell, Roslin; Ingle, Susan; Luccarini, Craig; Earl, Helena M; Pharoah, Paul PD; Dunning, Alison M; Caldas, Carlos

2012-05-30

307

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

308

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

309

Legionella pneumophila DNA in serum samples during Legionnaires' disease in relation to C-reactive protein levels.  

PubMed

Legionella pneumophila DNA can be detected in serum from patients with Legionnaires' disease (LD). We explored this observation studying the kinetics of L. pneumophila DNA in serum samples in relation to C-reactive protein (CRP). Eleven hospitalized patients with LD were studied. Diagnosis was made by Legionella urinary antigen test in 8 patients and seroconversion in 3 patients. A macrophage infectivity potentiator (MIP) real-time PCR was performed on 31 serum samples, including 20 follow-up serum samples. Serum samples obtained on the day of admission were MIP PCR-positive in 7 (64%) and MIP PCR-negative in 4 (36%) patients. Three (75%) of the 4 patients with a MIP PCR-negative serum sample on the day of admission became positive during follow-up. Overall, L. pneumophila DNA was detected in serum samples from 10 of the 11 patients (91%). CRP levels in the 7 patients with a positive MIP PCR serum sample on day of admission (499 +/- 144 mg/l; median +/- SD) were significantly higher than those in the 4 patients with a negative MIP PCR serum sample on the day of admission (244 +/- 97 mg/l). No difference in the severity of the disease on the day of admission was found between these patients. The presence of L. pneumophila DNA in serum is a common phenomenon in hospitalized patients with LD, although in some cases it is not yet present on the day of admission. L. pneumophila DNA in serum on the day of admission correlates with high CRP levels, but not with the severity of the disease. PMID:18855027

van de Veerdonk, F L; de Jager, C P C; Schellekens, J J A; Huijsmans, C J J; Beaumont, F; Hermans, M H A; Wever, P C

2009-04-01

310

Comparison of manual and automated DNA purification for measuring TREC in dried blood spot (DBS) samples with qPCR.  

PubMed

Automated nucleic acid extractions from dried blood spot (DBS) samples promises standardized sample treatment, low error rates, avoidance of contamination and requirement of less hands-on time. In the present study, non-automated and automated column based extraction processes using the QIAamp Investigator procedure were compared for the extraction of DNA from DBS samples. The concentration and the purity of DNA generated were determined by optical density readings. Furthermore qPCR downstream applications using the nucleic acids extracted with the two processes and albumin and T-cell receptor excision circles (TREC) copy numbers were measured and compared. The influence of the time of storage was also investigated by analyzing samples freshly dried and stored up to 11weeks at -20°C from the same individual. Finally, we provide arguments of preferentially choosing the automated procedure for extracting DNAs from DBS samples when downstream qPCR applications are required. PMID:22867745

Lang, Pierre-Olivier; Govind, Sheila; Dramé, Moustapha; Aspinall, Richard

2012-10-31

311

Damage response of XRCC1 at sites of DNA single strand breaks is regulated by phosphorylation and ubiquitylation after degradation of poly(ADP-ribose).  

PubMed

Single-strand breaks (SSBs) are the most common type of oxidative DNA damage and they are related to aging and many genetic diseases. The scaffold protein for repair of SSBs, XRCC1, accumulates at sites of poly(ADP-ribose) (pAR) synthesized by PARP, but it is retained at sites of SSBs after pAR degradation. How XRCC1 responds to SSBs after pAR degradation and how this affects repair progression are not well understood. We found that XRCC1 dissociates from pAR and is translocated to sites of SSBs dependent on its BRCTII domain and the function of PARG. In addition, phosphorylation of XRCC1 is also required for the proper dissociation kinetics of XRCC1 because (1) phosphorylation sites mutated in XRCC1 (X1 pm) cause retention of XRCC1 at sites of SSB for a longer time compared to wild type XRCC1; and (2) phosphorylation of XRCC1 is required for efficient polyubiquitylation of XRCC1. Interestingly, a mutant of XRCC1, LL360/361DD, which abolishes pAR binding, shows significant upregulation of ubiquitylation, indicating that pARylation of XRCC1 prevents the poly-ubiquitylation. We also found that the dynamics of the repair proteins DNA polymerase beta, PNK, APTX, PCNA and ligase I are regulated by domains of XRCC1. In summary, the dynamic damage response of XRCC1 is regulated in a manner that depends on modifications of polyADP-ribosylation, phosphorylation and ubiquitylation in live cells. PMID:23868975

Wei, Leizhen; Nakajima, Satoshi; Hsieh, Ching-Lung; Kanno, Shinichiro; Masutani, Mitsuko; Levine, Arthur S; Yasui, Akira; Lan, Li

2013-10-01

312

Damage response of XRCC1 at sites of DNA single strand breaks is regulated by phosphorylation and ubiquitylation after degradation of poly(ADP-ribose)  

PubMed Central

Summary Single-strand breaks (SSBs) are the most common type of oxidative DNA damage and they are related to aging and many genetic diseases. The scaffold protein for repair of SSBs, XRCC1, accumulates at sites of poly(ADP-ribose) (pAR) synthesized by PARP, but it is retained at sites of SSBs after pAR degradation. How XRCC1 responds to SSBs after pAR degradation and how this affects repair progression are not well understood. We found that XRCC1 dissociates from pAR and is translocated to sites of SSBs dependent on its BRCTII domain and the function of PARG. In addition, phosphorylation of XRCC1 is also required for the proper dissociation kinetics of XRCC1 because (1) phosphorylation sites mutated in XRCC1 (X1 pm) cause retention of XRCC1 at sites of SSB for a longer time compared to wild type XRCC1; and (2) phosphorylation of XRCC1 is required for efficient polyubiquitylation of XRCC1. Interestingly, a mutant of XRCC1, LL360/361DD, which abolishes pAR binding, shows significant upregulation of ubiquitylation, indicating that pARylation of XRCC1 prevents the poly-ubiquitylation. We also found that the dynamics of the repair proteins DNA polymerase beta, PNK, APTX, PCNA and ligase I are regulated by domains of XRCC1. In summary, the dynamic damage response of XRCC1 is regulated in a manner that depends on modifications of polyADP-ribosylation, phosphorylation and ubiquitylation in live cells. PMID:23868975

Wei, Leizhen; Nakajima, Satoshi; Hsieh, Ching-Lung; Kanno, Shinichiro; Masutani, Mitsuko; Levine, Arthur S.; Yasui, Akira; Lan, Li

2013-01-01

313

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

PubMed Central

The genus Azoarcus includes nitrogen-fixing, grass-associated strains as well as denitrying 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 Thauera 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 N2O 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.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7793946

Hurek, T; Reinhold-Hurek, B

1995-01-01

314

An alternative method for DNA extraction and PCR identification of Entamoeba histolytica and E. dispar in fecal samples.  

PubMed

Since it is known that Entamoeba dispar is non-pathogenic and morphologically similar to E. histolytica, there are many targets used in PCR for differentiating these species. However, obtaining high quality DNA from fecal samples is fundamental for PCR. Most methods are laborious or use kits that make diagnosis expensive. In the present work, a new simple, fast and cheap technique of DNA extraction from fecal samples was combined with a PCR for an episomal target in order to identify E. histolytica and E. dispar in feces. PMID:19486545

Vianna, E N; Costa, J O; Santos, C K S; Cury, M C; Silva, E F; Costa, A O; Gomes, M A

2009-06-01

315

Inferring separate parental admixture components in unknown DNA samples using autosomal SNPs  

PubMed Central

The identification of ancestral admixture proportions for human DNA samples has recently had success in forensic cases. Current methods infer admixture proportions for the target sample, but not for their parents, which provides an additional layer of information that may aid certain forensic investigations. We describe new maximum likelihood methods (LEAPFrOG and LEAPFrOG Expectation Maximisation), for inferring both an individual's admixture proportions and the admixture proportions possessed by the unobserved parents, with respect to two or more source populations, using single-nucleotide polymorphism data typed only in the target individual. This is achieved by examining the increase in heterozygosity in the offspring of parents who are from different populations or who represent different mixtures from a number of source populations. We validated the methods via simulation; combining chromosomes from different Hapmap Phase III population samples to emulate first-generation admixture. Performance was strong for individuals with mixed African/European (YRI/CEU) ancestry, but poor for mixed Japanese/Chinese (JPT/CHB) ancestry, reflecting the difficulty in distinguishing closely related source populations. A total of 11 African-American trios were used to compare the parental admixture inferred from their own genotypes against that inferred purely from their offspring genotypes. We examined the performance of 34 ancestry informative markers from a multiplex kit for ancestry inference. Simulations showed that estimates were unreliable when parents had similar admixture, suggesting more markers are needed. Our results demonstrate that ancestral backgrounds of case samples and their parents are obtainable to aid in forensic investigations, provided that high-throughput methods are adopted by the forensic community. PMID:22739346

Crouch, Daniel J M; Weale, Michael E

2012-01-01

316

Identification of Eukaryotic Open Reading Frames in Metagenomic cDNA Libraries Made from Environmental Samples  

PubMed Central

Here we describe the application of metagenomic technologies to construct cDNA libraries from RNA isolated from environmental samples. RNAlater (Ambion) was shown to stabilize RNA in environmental samples for periods of at least 3 months at ?20°C. Protocols for library construction were established on total RNA extracted from Acanthamoeba polyphaga trophozoites. The methodology was then used on algal mats from geothermal hot springs in Tengchong county, Yunnan Province, People's Republic of China, and activated sludge from a sewage treatment plant in Leicestershire, United Kingdom. The Tenchong libraries were dominated by RNA from prokaryotes, reflecting the mainly prokaryote microbial composition. The majority of these clones resulted from rRNA; only a few appeared to be derived from mRNA. In contrast, many clones from the activated sludge library had significant similarity to eukaryote mRNA-encoded protein sequences. A library was also made using polyadenylated RNA isolated from total RNA from activated sludge; many more clones in this library were related to eukaryotic mRNA sequences and proteins. Open reading frames (ORFs) up to 378 amino acids in size could be identified. Some resembled known proteins over their full length, e.g., 36% match to cystatin, 49% match to ribosomal protein L32, 63% match to ribosomal protein S16, 70% to CPC2 protein. The methodology described here permits the polyadenylated transcriptome to be isolated from environmental samples with no knowledge of the identity of the microorganisms in the sample or the necessity to culture them. It has many uses, including the identification of novel eukaryotic ORFs encoding proteins and enzymes. PMID:16391035

Grant, Susan; Grant, William D.; Cowan, Don A.; Jones, Brian E.; Ma, Yanhe; Ventosa, Antonio; Heaphy, Shaun

2006-01-01

317

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

318

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

319

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

320

The Viral Ubiquitin Ligase ICP0 Is neither Sufficient nor Necessary for Degradation of the Cellular DNA Sensor IFI16 during Herpes Simplex Virus 1 Infection  

PubMed Central

The cellular protein IFI16 colocalizes with the herpes simplex virus 1 (HSV-1) ubiquitin ligase ICP0 at early times of infection and is degraded as infection progresses. Here, we report that the factors governing the degradation of IFI16 and its colocalization with ICP0 are distinct from those of promyelocytic leukemia protein (PML), a well-characterized ICP0 substrate. Unlike PML, IFI16 colocalization with ICP0 was dependent on the ICP0 RING finger and did not occur when proteasome activity was inhibited. Expression of ICP0 in the absence of infection did not destabilize IFI16, the degradation occurred efficiently in the absence of ICP0 if infection was progressing efficiently, and IFI16 was relatively stable in wild-type (wt) HSV-1-infected U2OS cells. Therefore, IFI16 stability appears to be regulated by cellular factors in response to active HSV-1 infection rather than directly by ICP0. Because IFI16 is a DNA sensor that becomes associated with viral genomes during the early stages of infection, we investigated its role in the recruitment of PML nuclear body (PML NB) components to viral genomes. Recruitment of PML and hDaxx was less efficient in a proportion of IFI16-depleted cells, and this correlated with improved replication efficiency of ICP0-null mutant HSV-1. Because the absence of interferon regulatory factor 3 (IRF3) does not increase the plaque formation efficiency of ICP0-null mutant HSV-1, we speculate that IFI16 contributes to cell-mediated restriction of HSV-1 in a manner that is separable from its roles in IRF3-mediated interferon induction, but that may be linked to the PML NB response to viral infection. PMID:24089555

Cuchet-Lourenço, Delphine; Anderson, Gail; Sloan, Elizabeth; Orr, Anne

2013-01-01

321

Covalently Bound VirD2 Protein of Agrobacterium tumefaciens Protects the T-DNA from Exonucleolytic Degradation  

Microsoft Academic Search

We show that upon induction of Agrobacterium tumefaciens, free linear double-stranded T-DNA molecules as well as the previously described T-strands are generated from the Ti plasmid. A majority of these molecules are bound to a protein. We show that this protein is the product of the virulence gene virD2. This protein was found to be attached to the 5' terminus

Franz Durrenberger; Andreas Crameri; Barbara Hohn; Zdena Koukolikova-Nicola

1989-01-01

322

Human Origin Recognition Complex Large Subunit Is Degraded by Ubiquitin-Mediated Proteolysis after Initiation of DNA Replication  

Microsoft Academic Search

Eukaryotic cells possess overlapping mechanisms to ensure that DNA replication is restricted to the S phase of the cell cycle. The levels of hOrc1p, the largest subunit of the human origin recognition complex, vary during the cell division cycle. In rapidly proliferating cells, hOrc1p is expressed and targeted to chromatin as cells exit mitosis and prereplicative complexes are formed. Later,

Juan Méndez; X. Helena Zou-Yang; So-Young Kim; Masumi Hidaka; William P. Tansey; Bruce Stillman

2002-01-01

323

Piezoresistive microcantilever-based DNA sensor for sensitive detection of pathogenic Vibrio cholerae O1 in food sample.  

PubMed

Pathogenic Vibrio cholerae produces a cholera toxin which is the cause of a severe diarrheal disease called "Cholera". Available detection methods, including standard bacteriological test and immuno-based detection, are specific to the suspected pathogenic V. cholerae O1 and O139, but they are not specific to the cholera toxin producible strain. This work combined the polymerase chain reaction (PCR) of cholera toxin gene, ctxA gene, and microcantilever-based DNA sensor to improve the sensitivity and specificity of detection. Gold coated microcantilever, 250 µm long and 50 µm wide, with an embedded polysilicon wire acting as a piezoresistive material was modified by a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) for immobilization of specific DNA probe via avidin layer on the surface. The avidin and 5' biotinylated single-stranded DNA (ssDNA) probe concentrations were optimized for the immobilization at 50 µg/mL and 1 µM, respectively. The hybridization between ssDNA probe on this DNA sensor and target DNA creates nanomechanical bending and resistance change of piezoresistive material inside the beam. This microcantilever-based DNA sensor offers a detection sensitivity of 3.25 pg or 14 nM of DNA template for ctxA gene detection. The lowest number of V. cholerae O1 in food sample with and without the enrichment process that the polymerase chain reaction (PCR) for ctxA gene combined with this DNA sensor can detect is 0.835 and 835 cells/g, respectively. This detection sensitivity is 10 times higher than that of the conventional PCR method. PMID:25113053

Khemthongcharoen, Numfon; Wonglumsom, Wijit; Suppat, Assawapong; Jaruwongrungsee, Kata; Tuantranont, Adisorn; Promptmas, Chamras

2015-01-15

324

Upscaled CTAB-based DNA extraction and real-time PCR assays for Fusarium culmorum and F. graminearum DNA in plant material with reduced sampling error.  

PubMed

Fusarium graminearum Schwabe (Gibberella zeae Schwein. Petch.) and F. culmorum W.G. Smith are major mycotoxin producers in small-grain cereals afflicted with Fusarium head blight (FHB). Real-time PCR (qPCR) is the method of choice for species-specific, quantitative estimation of fungal biomass in plant tissue. We demonstrated that increasing the amount of plant material used for DNA extraction to 0.5-1.0 g considerably reduced sampling error and improved the reproducibility of DNA yield. The costs of DNA extraction at different scales and with different methods (commercial kits versus cetyltrimethylammonium bromide-based protocol) and qPCR systems (doubly labeled hybridization probes versus SYBR Green) were compared. A cost-effective protocol for the quantification of F. graminearum and F. culmorum DNA in wheat grain and maize stalk debris based on DNA extraction from 0.5-1.0 g material and real-time PCR with SYBR Green fluorescence detection was developed. PMID:19330077

Brandfass, Christoph; Karlovsky, Petr

2008-11-01

325

DNA detection of Toxoplasma gondii in sheep milk and blood samples in relation to phase of infection.  

PubMed

Toxoplasma gondii is a worldwide parasite that is important both for veterinary medicine (economic losses in the herd) and for public health (immunocompromised patients and pregnant women). An important source of Toxoplasma infection in humans is consumption of contaminated meat and milk (undercooked meat and unpasteurized milk). Small ruminants are important in both milk and meat production throughout the world because of free-range husbandry. The purpose of our study was to detect the presence of T. gondii DNA in ewes' milk 1 month after the term, and to determine the relationship between the occurrence of this DNA in blood and milk based on the phase of infection. Using enzyme-linked immunosorbent assay (ELISA) the animals were divided into two groups (immunoglobulin M positive (IgM+), IgM-). With real-time polymerase chain reaction (PCR), T. gondii DNA was detected in seven milk samples (28%) and five blood samples (20%) of the IgM+ group (25 samples). In the IgM- group T. gondii DNA was detected in two milk samples (3.6%) out of 55 samples. PMID:25630551

Luptakova, L; Benova, K; Rencko, A; Petrovova, E

2015-03-15

326

Development of a Rapid and Efficient Method for Non-Lethal DNA Sampling and Genotyping in Scallops  

PubMed Central

Non-lethal DNA sampling has long appealed to researchers studying population and conservation genetics, as it does not necessitate removing individuals permanently from their natural environment or destroying valuable samples. However, such an approach has not yet been well established in bivalves. In this study, we demonstrate that the gill represents a good source of tissue for non-lethal sampling in scallops. Removal of a few gill filaments caused no noticeable behavioral abnormalities or increased mortality rates in Zhikong scallop (Chlamys farreri) during a three-month period of observation. To facilitate rapid gill-based DNA extraction, six methods (MA-MF) were designed and evaluated, each requiring less than one hour of processing time. The optimal method was identified as MF, in terms of maintaining DNA integrity and genotyping accuracy. Further optimization of MF method by orthogonal experimental design suggested that the utilization of gills could be limited to 2 mg of sample, which is sufficient for performing up to 20,000 PCR reactions. We also demonstrate the excellent cross-species utility of MF in two additional scallop species, Yesso scallop (Patinopecten yessoensis) and bay scallop (Argopecten irradians). Taken together, our study provides a rapid and efficient approach for applying non-lethal DNA sampling in bivalve species, which would serve as a valuable tool for maintaining bivalve populations and conservation genetics, as well as in breeding studies. PMID:23874509

Miao, Yan; Sun, Changsen; Hu, Liping; Zhang, Ru; Fu, Xiaoteng; Zhang, Lingling; Hu, Xiaoli; Wang, Shi; Bao, Zhenmin

2013-01-01

327

DNA analysis using an integrated microchip for multiplex PCR amplification and electrophoresis for reference samples.  

PubMed

A system that automatically performs the PCR amplification and microchip electrophoretic (ME) separation for rapid forensic short tandem repeat (STR) forensic profiling in a single disposable plastic chip is demonstrated. The microchip subassays were optimized to deliver results comparable to conventional benchtop methods. The microchip process was accomplished in sub-90 min compared with >2.5 h for the conventional approach. An infrared laser with a noncontact temperature sensing system was optimized for a 45 min PCR compared with the conventional 90 min amplification time. The separation conditions were optimized using LPA-co-dihexylacrylamide block copolymers specifically designed for microchip separations to achieve accurate DNA size calling in an effective length of 7 cm in a plastic microchip. This effective separation length is less than half of other reports for integrated STR analysis and allows a compact, inexpensive microchip design. This separation quality was maintained when integrated with microchip PCR. Thirty samples were analyzed conventionally and then compared with data generated by the microfluidic chip system. The microfluidic system allele calling was 100% concordant with the conventional process. This study also investigated allelic ladder consistency over time. The PCR-ME genetic profiles were analyzed using binning palettes generated from two sets of allelic ladders run three and six months apart. Using these binning palettes, no allele calling errors were detected in the 30 samples demonstrating that a microfluidic platform can be highly consistent over long periods of time. PMID:25091472

Le Roux, Delphine; Root, Brian E; Reedy, Carmen R; Hickey, Jeffrey A; Scott, Orion N; Bienvenue, Joan M; Landers, James P; Chassagne, Luc; de Mazancourt, Philippe

2014-08-19

328

Five commercial DNA extraction systems tested and compared on a stool sample collection.  

PubMed

In this study, 5 different commercial DNA extraction systems were tested on a stool sample collection containing 81 clinical stool specimens that were culture-positive for diarrheagenic Escherichia coli, Campylobacter jejuni, Salmonella enterica, or Clostridium difficile. The purified DNAs were analyzed by polymerase chain reaction (PCR) directed toward the relevant organisms. The results showed that conventional PCR combined with the extraction systems BioRobot EZ1 (Qiagen, Hilden, Germany), Bugs'n Beads (Genpoint, Oslo, Norway), ChargeSwitch (Invitrogen, Paisley, UK), QIAamp Stool Mini Kit (Qiagen), and 2 protocols (generic and Specific A) for EasyMag (BioMérieux, Marcy I'Etoile, France) were able to identify 89%, 62%, 85%, 88%, 85%, and 91%, respectively, of the pathogens originally identified by conventional culture-based methods. When TaqMan PCR was combined with the EasyMag Specific A protocol, 99% of the samples were correctly identified. The results demonstrate that the extraction efficiencies can vary significantly among different extraction systems, careful optimization may have a significant positive effect, and the use of sensitive and specific detection methods like TaqMan PCR is an ideal choice for this type of analysis. PMID:21353945

Persson, Søren; de Boer, Richard F; Kooistra-Smid, Anna M D; Olsen, Katharina E P

2011-03-01

329

NKLP27: A Teleost NK-Lysin Peptide that Modulates Immune Response, Induces Degradation of Bacterial DNA, and Inhibits Bacterial and Viral Infection  

PubMed Central

NK-lysin is an antimicrobial protein produced by cytotoxic T lymphocytes and natural killer cells. In this study, we examined the biological property of a peptide, NKLP27, derived from tongue sole (Cynoglossus semilaevis) NK-lysin. NKLP27 is composed of 27 amino acids and shares little sequence identity with known NK-lysin peptides. NKLP27 possesses bactericidal activity against both Gram-negative and Gram-positive bacteria including common aquaculture pathogens. The bactericidal activity of NKLP27 was dependent on the C-terminal five residues, deletion of which dramatically reduced the activity of NKLP27. During its interaction with the target bacterial cells, NKLP27 destroyed cell membrane integrity, penetrated into the cytoplasm, and induced degradation of genomic DNA. In vivo study showed that administration of tongue sole with NKLP27 before bacterial and viral infection significantly reduced pathogen dissemination and replication in tissues. Further study revealed that fish administered with NKLP27 exhibited significantly upregulated expression of the immune genes including those that are known to be involved in antibacterial and antiviral defense. These results indicate that NKLP27 is a novel antimicrobial against bacterial and viral pathogens, and that the observed effect of NKLP27 on bacterial DNA and host gene expression adds new insights to the action mechanism of fish antimicrobial peptides. PMID:25180858

Sun, Li

2014-01-01

330

DNA isolation from dry and fresh samples of polysaccharide-rich plants  

Microsoft Academic Search

DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation\\u000a procedures and downstream applications such as DNA restriction, amplification, and cloning. The chemotypic heterogeneity among\\u000a species may not permit optimal DNA yield with a single protocol; thus, even closely related species may require different\\u000a isolating protocols. Here we describe a

Arun Dev Sharma; Prabhjot Kaur Gill; Prabhjeet Singh

2002-01-01

331

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

E-print Network

to valuable specimens, we describe a nondestructive method for extracting DNA from ancient human remains methods have been developed for extracting aDNA from skeletal remains, virtually all pro- tocols require extraction; mtDNA; autosomal STR genotyping ABSTRACT Museum curators and living commun- ities are sometimes

Kemp, Brian M.

332

Concerted Action of the Ubiquitin-Fusion Degradation Protein 1 (Ufd1) and Sumo-Targeted Ubiquitin Ligases (STUbLs) in the DNA-Damage Response  

PubMed Central

In eukaryotes many players in the DNA-damage response (DDR) catalyze protein sumoylation or ubiquitylation. Emphasis has been placed on how these modifications orchestrate the sequential recruitment of repair factors to sites of DNA damage or stalled replication forks. Here, we shed light on a pathway in which sumoylated factors are eliminated through the coupled action of Sumo-targeted ubiquitin ligases (STUbLs) and the ubiquitin-fusion degradation protein 1 (Ufd1). Ufd1 is a subunit of the Cdc48-Ufd1-Npl4 complex implicated in the sorting of ubiquitylated substrates for degradation by the proteasome. We find that in fission yeast, Ufd1 interacts physically and functionally with the Sumo-targeted ubiquitin ligase (STUbL) Rfp1, homologous to human RNF4, and with the Sumo E3 ligase Pli1, homologous to human PIAS1. Deleting a C-terminal domain of Ufd1 that mediates the interaction of Ufd1 with Rfp1, Pli1, and Sumo (ufd1?Ct213-342) lead to an accumulation of high-molecular-weight Sumo conjugates and caused severe genomic instabilities. The spectrum of sensitivity of ufd1?Ct213-342 cells to genotoxins, the epistatic relationships of ufd1?Ct213-342 with mutations in DNA repair factors, and the localization of the repair factor Rad22 in ufd1?Ct213-342 cells point to ufd1?Ct213-342 cells accumulating aberrant structures during replication that require homologous recombination (HR) for their repair. We present evidence that HR is however often not successful in ufd1?Ct213-342 cells and we identify Rad22 as one of the high-molecular-weight conjugates accumulating in the ufd1?Ct213-342 mutant consistent with Rad22 being a STUbL/Ufd1 substrate. Suggesting a direct role of Ufd1 in the processing of Sumo-conjugates, Ufd1 formed nuclear foci colocalizing with Sumo during the DDR, and Sumo-conjugates accumulated in foci in the ufd1?Ct213-342 mutant. Broader functional relationships between Ufd1 and STUbLs conceivably affect numerous cellular processes beyond the DDR. PMID:24265825

Køhler, Julie Bonne; Jørgensen, Maria Louise Mønster; Beinoraité, Gabriele; Thorsen, Michael; Thon, Geneviève

2013-01-01

333

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

334

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

335

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

PubMed

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

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

2014-06-01

336

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

337

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

338

Probe-Directed Degradation (PDD) for Flexible Removal of Unwanted cDNA Sequences from RNA-Seq Libraries.  

PubMed

Most applications for RNA-seq require the depletion of abundant transcripts to gain greater coverage of the underlying transcriptome. The sequences to be targeted for depletion depend on application and species and in many cases may not be supported by commercial depletion kits. This unit describes a method for generating RNA-seq libraries that incorporates probe-directed degradation (PDD), which can deplete any unwanted sequence set, with the low-bias split-adapter method of library generation (although many other library generation methods are in principle compatible). The overall strategy is suitable for applications requiring customized sequence depletion or where faithful representation of fragment ends and lack of sequence bias is paramount. We provide guidelines to rapidly design specific probes against the target sequence, and a detailed protocol for library generation using the split-adapter method including several strategies for streamlining the technique and reducing adapter dimer content. © 2015 by John Wiley & Sons, Inc. PMID:25827346

Archer, Stuart K; Shirokikh, Nikolay E; Preiss, Thomas

2015-01-01

339

Comparative analysis of microbiome between accurately identified 16S rDNA and quantified bacteria in simulated samples.  

PubMed

Although 16S rRNA gene (rDNA) sequencing is the gold standard for categorizing bacteria or characterizing microbial communities its clinical utility is limited by bias in metagenomic studies, in either the experiments or the data analyses. To evaluate the efficiency of current metagenomic methods, we sequenced seven simulated samples of ten bacterial species mixed at different concentrations. The V3 region of 16S rDNA was targeted and used to determine the distribution of bacterial species. The number of target sequences in individual simulated samples was in the range 1-1000 to provide a better reflection of natural microbial communities. However, for a given bacterial species present in the same proportion but at different concentrations, the observed percentage of 16S rDNAs was similar, except at very low concentrations that cannot be detected by real-time PCR. These results confirmed that the comparative microbiome in a sample characterized by 16S rDNA sequencing is sufficient to detect not only potential infectious pathogens, but also the relative proportion of 16S rDNA in the sample. PMID:24293637

Wang, Haiyin; Du, Pengcheng; Li, Juan; Zhang, Yuanyuan; Zhang, Wen; Han, Na; Woo, Patrick C Y; Chen, Chen

2014-03-01

340

Comparison of DNA Extraction Methods for Microbial Community Profiling with an Application to Pediatric Bronchoalveolar Lavage Samples  

Microsoft Academic Search

Barcoded amplicon sequencing is rapidly becoming a standard method for profiling microbial communities, including the human respiratory microbiome. While this approach has less bias than standard cultivation, several steps can introduce variation including the type of DNA extraction method used. Here we assessed five different extraction methods on pediatric bronchoalveolar lavage (BAL) samples and a mock community comprised of nine

Dana Willner; Joshua Daly; David Whiley; Keith Grimwood; Claire E. Wainwright; Philip Hugenholtz

2012-01-01

341

Mitochondrial DNA Marker EST00083 Is Not Associated with High vs. Average IQ in a German Sample.  

ERIC Educational Resources Information Center

Tested the association of a mitochondrial DNA marker (EST00083) with high IQ in a sample of 47 German adults with high IQ scores and 77 adults with IQs estimated at lower than 110. Results do not support the hypothesis that high IQ is associated with this marker. (SLD)

Moises, Hans W.; Yang, Liu; Kohnke, Michael; Vetter, Peter; Neppert, Jurgen; Petrill, Stephen A.; Plomin, Robert

1998-01-01

342

The Anaphase Promoting Complex Regulates Yeast Lifespan and rDNA Stability by Targeting Fob1 for Degradation  

PubMed Central

Genomic stability, stress response, and nutrient signaling all play critical, evolutionarily conserved roles in lifespan determination. However, the molecular mechanisms coordinating these processes with longevity remain unresolved. Here we investigate the involvement of the yeast anaphase promoting complex (APC) in longevity. The APC governs passage through M and G1 via ubiquitin-dependent targeting of substrate proteins and is associated with cancer and premature aging when defective. Our two-hybrid screen utilizing Apc5 as bait recovered the lifespan determinant Fob1 as prey. Fob1 is unstable specifically in G1, cycles throughout the cell cycle in a manner similar to Clb2 (an APC target), and is stabilized in APC (apc5CA) and proteasome (rpn10?) mutants. Deletion of FOB1 increased replicative lifespan (RLS) in wild type (WT), apc5CA, and apc10? cells, and suppressed apc5CA cell cycle progression and rDNA recombination defects. Alternatively, increased FOB1 expression decreased RLS in WT cells, but did not reduce the already short apc5CA RLS, suggesting an epistatic interaction between apc5CA and fob1?. Mutation to a putative L-Box (Fob1E420V), a Destruction Box-like motif, abolished Fob1 modifications, stabilized the protein, and increased rDNA recombination. Our work provides a mechanistic role played by the APC to promote replicative longevity and genomic stability in yeast. PMID:24361936

Menzel, Johannes; Malo, Mackenzie E.; Chan, Cynthia; Prusinkiewicz, Martin; Arnason, Terra G.; Harkness, Troy A. A.

2014-01-01

343

Electron-induced degradation of 8-bromo-2'-deoxyadenosine 3',5'-diphosphate, a DNA radiosensitizing nucleotide.  

PubMed

The phosphodiester bond cleavage in 8-bromo-2'-deoxyadenosine 3',5'-diphosphate (BrdADP), as a model of electron induced single strand break (SSB) in labeled DNA, was investigated at the B3LYP/6-31++G(d,p) level of theory both in the gas phase and in water solution. Barrier-free and highly exergonic, especially in water solution (-2.83 eV), release of the bromide anion due to electron attachment confirms radiosensitizing properties of 8-bromoadenine. Thermodynamically (-19 kcal/mol) and kinetically (barrier of 10-13 kcal/mol) feasible hydrogen atom transfer from the C3' or C5' sites of the deoxyribose moiety to the C8 center of adenine radical is followed by a relatively low (14-18 kcal/mol) activation barrier O-P bond cleavage at either the 3'- or the 5'-site. The C5' radical may also stabilize via the formation of 5',8-cycloadenosine. The latter process has favorable thermodynamic and kinetic characteristics, which makes the O-P bond breakage at the 5'-site highly unlikely. Thus, the O-P cleavage reaction, being an equivalent of SSB in DNA labeled with 8-bromoadenine, should lead to the formation of cyclic ketone, which if identified in a radiolytic experiment, would confirm the mechanism proposed in the current study. PMID:23802987

Chomicz, Lidia; Leszczynski, Jerzy; Rak, Janusz

2013-07-25

344

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

345

Accurate measurement of circulating mitochondrial DNA content from human blood samples using real-time quantitative PCR.  

PubMed

We describe a protocol to accurately measure the amount of human mitochondrial DNA (MtDNA) in peripheral blood samples which can be modified to quantify MtDNA from other body fluids, human cells, and tissues. This protocol is based on the use of real-time quantitative PCR (qPCR) to quantify the amount of MtDNA relative to nuclear DNA (designated the Mt/N ratio). In the last decade, there have been increasing numbers of studies describing altered MtDNA or Mt/N in circulation in common nongenetic diseases where mitochondrial dysfunction may play a role (for review see Malik and Czajka, Mitochondrion 13:481-492, 2013). These studies are distinct from those looking at genetic mitochondrial disease and are attempting to identify acquired changes in circulating MtDNA content as an indicator of mitochondrial function. However, the methodology being used is not always specific and reproducible. As more than 95 % of the human mitochondrial genome is duplicated in the human nuclear genome, it is important to avoid co-amplification of nuclear pseudogenes. Furthermore, template preparation protocols can also affect the results because of the size and structural differences between the mitochondrial and nuclear genomes. Here we describe how to (1) prepare DNA from blood samples; (2) pretreat the DNA to prevent dilution bias; (3) prepare dilution standards for absolute quantification using the unique primers human mitochondrial genome forward primer (hMitoF3) and human mitochondrial genome reverse primer(hMitoR3) for the mitochondrial genome, and human nuclear genome forward primer (hB2MF1) and human nuclear genome reverse primer (hB2MR1) primers for the human nuclear genome; (4) carry out qPCR for either relative or absolute quantification from test samples; (5) analyze qPCR data; and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use. PMID:25631009

Ajaz, Saima; Czajka, Anna; Malik, Afshan

2015-01-01

346

NOCIt: A computational method to infer the number of contributors to DNA samples analyzed by STR genotyping.  

PubMed

Repetitive sequences in the human genome called short tandem repeats (STRs) are used in human identification for forensic purposes. Interpretation of DNA profiles generated using STRs is often problematic because of uncertainty in the number of contributors to the sample. Existing methods to identify the number of contributors work on the number of peaks observed and/or allele frequencies. We have developed a computational method called NOCIt that calculates the a posteriori probability (APP) on the number of contributors. NOCIt works on single source calibration data consisting of known genotypes to compute the APP for an unknown sample. The method takes into account signal peak heights, population allele frequencies, allele dropout and stutter-a commonly occurring PCR artifact. We tested the performance of NOCIt using 278 experimental and 40 simulated DNA mixtures consisting of one to five contributors with total DNA mass from 0.016 to 0.25ng. NOCIt correctly identified the number of contributors in 83% of the experimental samples and in 85% of the simulated mixtures, while the accuracy of the best pre-existing method to determine the number of contributors was 72% for the experimental samples and 73% for the simulated mixtures. Moreover, NOCIt calculated the APP for the true number of contributors to be at least 1% in 95% of the experimental samples and in all the simulated mixtures. PMID:25625964

Swaminathan, Harish; Grgicak, Catherine M; Medard, Muriel; Lun, Desmond S

2015-05-01

347

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

348

An Improved Protocol for DNA Extraction from Alkaline Soil and Sediment Samples for Constructing Metagenomic Libraries  

Microsoft Academic Search

An improved single-step protocol has been developed for extracting pure community humic substance-free DNA from alkaline soils\\u000a and sediments. The method is based on direct cell lysis in the presence of powdered activated charcoal and polyvinylpolypyrrolidone\\u000a followed by precipitation with polyethyleneglycol and isopropanol. The strategy allows simultaneous isolation and purification\\u000a of DNA while minimizing the loss of DNA with respect

Digvijay Verma; T. Satyanarayana

349

Affinity Purification of DNA and RNA from Environmental Samples with Peptide Nucleic Acid Clamps  

Microsoft Academic Search

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sed- iment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO4, 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate (SDS)) a PNA clamp recovered significantly more

DARRELL P. CHANDLER; JENNIE R. STULTS; SHARON CEBULA; BEATRICE L. SCHUCK; DEREK W. WEAVER; KEVIN K. ANDERSON; MICHAEL EGHOLM; FRED J. BROCKMAN

2000-01-01

350

Application of a convenient DNA extraction method and multiplex PCR for the direct detection of Staphylococcus aureus and Yersinia enterocolitica in milk samples  

Microsoft Academic Search

The application of PCR for the direct and sensitive detection of food-borne pathogens is largely affected by the quality of the template DNA prepared from food samples. In the present study, a chemical extraction method of bacterial DNA from spiked milk samples for the direct detection of Staphylococcus aureus and Yersinia enterocolitica was evaluated by PCR. Gene specific primers were

A. Ramesh; B. P. Padmapriya; A. Chrashekar; M. C. Varadaraj

2002-01-01

351

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

352

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.

2015-01-01

353

Degradation Scheme graceful degradation  

E-print Network

degradation scheme OS degradation scheme OS Solaris Linux FreeBSD Windows Server Linux 3 OS Tomcat Solaris OS # of concurrent requests (Heavy) 0 1 2 3 4 Requests/sec(Heavy) (a) Solaris 9 (b) FreeBSD 5.2.1 (c) Windows 2003 Tomcat Solaris 9 Linux 2.6.7 2.6.5 2.4.18 FreeBSD 5.2.1 Windows 2003 Server Enterprise Edition Xeon 3

Chiba, Shigeru

354

RFLP analysis of forensic DNA samples with single-locus VNTR genetic markers.  

PubMed

This unit covers the many and varied methods for extracting DNA from such diverse specimens as blood, tissue, stamps and envelopes, and cigarette butts, among others. Modifications to the methods that allow the DNA to be used for either PCR or Southern blotbased analyses are also included. PMID:18428262

Bing, D H; Bieber, F R

2001-05-01

355

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

356

Assessment of DNA encapsulation, a new room-temperature DNA storage method.  

PubMed

A new procedure for room-temperature storage of DNA was evaluated whereby DNA samples from human tissue, bacteria, and plants were stored under an anoxic and anhydrous atmosphere in small glass vials fitted in stainless-steel, laser-sealed capsules (DNAshells(®)). Samples were stored in DNAshells(®) at room temperature for various periods of time to assess any degradation and compare it to frozen control samples and those stored in GenTegra™ tubes. The study included analysis of the effect of accelerated aging by using a high temperature (76°C) at 50% relative humidity. No detectable DNA degradation was seen in samples stored in DNAshells(®) at room temperature for 18 months. Polymerase chain reaction experiments, pulsed field gel electrophoresis, and amplified fragment length polymorphism analyses also demonstrated that the protective properties of DNAshells(®) are not affected by storage under extreme conditions (76°C, 50% humidity) for 30 hours, guaranteeing 100 years without DNA sample degradation. However, after 30 hours of storage at 76°C, it was necessary to include adjustments to the process in order to avoid DNA loss. Successful protection of DNA was obtained for 1 week and even 1 month of storage at high temperature by adding trehalose, which provides a protective matrix. This study demonstrates the many advantages of using DNAshells(®) for room-temperature storage, particularly in terms of long-term stability, safety, transport, and applications for molecular biology research. PMID:24955733

Clermont, Dominique; Santoni, Sylvain; Saker, Safa; Gomard, Maite; Gardais, Eliane; Bizet, Chantal

2014-06-01

357

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

2015-01-01

358

Glycogen synthase kinase (GSK) 3? phosphorylates and protects nuclear myosin 1c from proteasome-mediated degradation to activate rDNA transcription in early G1 cells.  

PubMed

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

2014-06-01

359

DNA degradation by bleomycin: evidence for 2'R-proton abstraction and for C-O bond cleavage accompanying base propenal formation.  

PubMed

Reaction of poly(dA-[2'S-3H]dU) with activated bleomycin yields [3H]uracil propenal that completely retains the tritium label. In contrast, we have previously shown that reaction of poly(dA-[2'R-3H]dU) with activated bleomycin affords unlabeled uracil propenal [Wu, J. C., Kozarich, J. W., & Stubbe, J. (1983) J. Biol. Chem. 258, 4694-4697]. We have also prepared both cis- and trans-thymine propenals by chemical synthesis and have observed that the trans isomer is the exclusive product of the bleomycin reaction. Moreover, the cis isomer was found to be stable to the conditions of bleomycin-induced DNA degradation. Taken together, these results establish that the formation of trans-uracil propenal occurs via an anti-elimination mechanism with the stereospecific abstraction of the 2'R proton. The question of phosphodiester bond cleavage during base propenal formation has also been addressed by the analysis of the fate of oxygen-18 in poly(dA-[3'-18O]dT) upon reaction with activated bleomycin. The 5'-monophosphate oligonucleotide ends produced from thymine propenal formation have been converted to inorganic phosphate by the action of alkaline phosphatase, and the phosphate has been analyzed for 18O content by 31P NMR spectroscopy. The oxygen-18 is retained in the inorganic phosphate, establishing that the formation of thymine propenal by activated bleomycin proceeds with C-O bond cleavage at the 3'-position. PMID:2431710

Ajmera, S; Wu, J C; Worth, L; Rabow, L E; Stubbe, J; Kozarich, J W

1986-10-21

360

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

361

Application of the BioMek 2000 Laboratory Automation Workstation and the DNA IQ System to the extraction of forensic casework samples.  

PubMed

Robotic systems are commonly utilized for the extraction of database samples. However, the application of robotic extraction to forensic casework samples is a more daunting task. Such a system must be versatile enough to accommodate a wide range of samples that may contain greatly varying amounts of DNA, but it must also pose no more risk of contamination than the manual DNA extraction methods. This study demonstrates that the BioMek 2000 Laboratory Automation Workstation, used in combination with the DNA IQ System, is versatile enough to accommodate the wide range of samples typically encountered by a crime laboratory. The use of a silica coated paramagnetic resin, as with the DNA IQ System, facilitates the adaptation of an open well, hands off, robotic system to the extraction of casework samples since no filtration or centrifugation steps are needed. Moreover, the DNA remains tightly coupled to the silica coated paramagnetic resin for the entire process until the elution step. A short pre-extraction incubation step is necessary prior to loading samples onto the robot and it is at this step that most modifications are made to accommodate the different sample types and substrates commonly encountered with forensic evidentiary samples. Sexual assault (mixed stain) samples, cigarette butts, blood stains, buccal swabs, and various tissue samples were successfully extracted with the BioMek 2000 Laboratory Automation Workstation and the DNA IQ System, with no evidence of contamination throughout the extensive validation studies reported here. PMID:14979341

Greenspoon, Susan A; Ban, Jeffrey D; Sykes, Karen; Ballard, Elizabeth J; Edler, Shelley S; Baisden, Melissa; Covington, Brian L

2004-01-01

362

Mitochondrial DNA Typing Screens with Control Region and Coding Region SNPs  

Microsoft Academic Search

Mitochondrial DNA (mtDNA) analysis has found an important niche in forensic DNA typing. It is used with highly degraded samples or low-copy number materials such as might be found from shed hair or bones exposed to severe environmental conditions. The primary advantage of mtDNA is that it is present in high copy number within cells and therefore more likely to

Margaret C. Kline; Peter M. Vallone; Janette W. Redman; David L. Duewer; Cassandra D. Calloway; John M. Butler

2005-01-01

363

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

364

The Shape Parameter of Liposomes and DNA-Lipid Complexes Determined by Viscometry Utilizing Small Sample Volumes  

Microsoft Academic Search

A minicapillary viscometer utilizing <0.5ml of sample at a volume fraction of <0.1% is described. The calculated a\\/b of DPPC\\/DPPG multilamellar liposome was 1.14 as prolate ellipsoids and a\\/b of dioleoylpropyltrimethyl ammonium methylsulfate-DNA complex at a charge ratio of 4:1 (+\\/?) was 3.7 as prolate ellipsoids or 4.9 as oblate ellipsoids. The deviation of shape from perfect sphere is thus

Y. Sun; X. Li; N. Düzgüne??; Y. Takaoka; S. Ohi; S. Hirota

2003-01-01

365

Genotyping of Chlamydia trachomatis strains from culture and clinical samples using an ompA-based DNA microarray assay  

Microsoft Academic Search

Current typing methods of Chlamydia (C.) trachomatis are mainly based on the diversity of the ompA gene, which is coding for the major outer membrane protein A. The present study aimed at facilitating genotyping of strains of this obligate intracellular human pathogen by developing a DNA microarray assay using the ArrayTube™ format for individual samples and the ArrayStrip™ format for

Anke Ruettger; Jens Feige; Peter Slickers; Evelyn Schubert; Servaas A. Morré; Yvonne Pannekoek; Björn Herrmann; Henry J. C. de Vries; Ralf Ehricht; Konrad Sachse

2011-01-01

366

Rapid method for screening dried blood samples on filter paper for human immunodeficiency virus type 1 DNA.  

PubMed

PCR is a highly sensitive method for the detection of human immunodeficiency virus type 1 (HIV-1) nucleic acids in blood mononuclear cells and plasma. However, blood separation techniques require extensive laboratory support systems and are difficult when a limited volume of blood is available, which is often the case for infants. The use of blood samples stored on filter paper has many advantages for the detection of perinatal HIV-1 infection, but current methods require extraction and purification of target DNA prior to PCR amplification. We report a highly sensitive and rapid method for the extraction and detection of HIV-1 DNA in infant blood samples stored on filter papers. Because this rapid protocol does not involve steps for the removal of potential inhibitors of the PCR, the highest sensitivity is achieved by testing the filter paper lysate in quadruplicate. Assays for HIV-1 DNA were done by using nested PCR techniques that amplify HIV-1 gag DNA from blood spot samples on filter paper and from corresponding viably frozen mononuclear cells separated from venous blood samples obtained from 111 infants born to HIV-1-seropositive mothers. PCR results with blood from filter papers showed 100% specificity (95% confidence internal [CI] 93.1 to 100%) and 96% (95% CI, 88.65 to 98.9%) and 88% (95% CI, 79.2 to 94.5%) sensitivity (for quadruplicate and duplicate tests, respectively) compared to PCR results with blood mononuclear cells. Moreover, this method could detect HIV-1 sequences of multiple subtypes. PMID:9889216

Panteleeff, D D; John, G; Nduati, R; Mbori-Ngacha, D; Richardson, B; Kreiss, J; Overbaugh, J

1999-02-01

367

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

368

DNA . DNA  

E-print Network

-base non 1-Base non Watson-Crick DNA Hybridization Simulation O O DNA Hybridization SimulationIntelligence Lab. School of Computer Science and Engineering, Seoul National University 1-Base non Watson-Crick DNA DNA . #12;Watson-Crick base pair 1-base dangling end hybridization . PCR

369

DNA Microarray  

NSDL National Science Digital Library

This animated YouTube video, created by Southwest Center for Microsystems Education (SCME), illustrates how DNA microarrays work in the context of nanofabrication. The animation and associated narration describe "how a DNA microarray identifies complementary DNA (cDNA) strands from a sample. On the substrate of the array are synthetic ssDNA stands or oligonucleotides (oligos). Each grid in the microarray contains hundreds to thousands of oligos with a unique DNA sequence. These oligos hybridize with cDNA from one of two samples. There are thousands of grids on the array allowing for the simultaneous identification of thousands of different DNA sequences. Each sample cDNA is "tagged" with the red or green tag as shown in the animation. The "tags" enable an interpretation of the DNA hybridizations." A supporting learning module and activities can be downloaded from the SCME website.

370

Localized Nanoscopic Surface Measurements of Nickel-Modified Mica for Single Molecule DNA Sequence Sampling  

PubMed Central

Cleaved, cation-derivatized Muscovite mica is utilized extensively in AFM imaging due to its flatness over large areas (millimeter cleavage planes with local RMS roughness <0.3 nm), ease of preparation and ability to adsorb charged biomolecules such as DNA1–3. In particular, NiCl2 treatment has become a common method for controlling DNA adsorption on mica substrates while retaining the mica’s ultra flat surface4. While several studies have modeled the mica:metal-ion:DNA system using macroscopic colloidal theory (DLVO, etc) 4–8, Ni-mica’s physicochemical properties have not been well characterized on the nanoscale. Efforts to manipulate and engineer DNA nanostructures would benefit greatly from a better understanding of the surface chemistry of Ni:mica. Here we present in-situ, nanometer- and attogram-scale measurements, and thermodynamic simulation results that show the surface chemistry of nickel treated mica is more complex than generally appreciated by AFM practitioners, due to metal ion speciation effects present at neutral pH. We also show that under certain preparations, Ni:Mica allows in situ, nanoscopic nucleotide sequence mapping within individual surface-adsorbed DNA molecules by permitting localized, controlled desorption of the double helix by soluble DNA binding enzymes. These results should aid efforts to precisely control DNA:mica binding affinity, particularly at physiological pH ranges required by enzymatic biochemistry (pH 7.0–8.5)4–8, and facilitate development of more complex and useful biochemical manipulations of adsorbed DNA, such as single molecule sequencing. PMID:21033675

Hsueh, Carlin; Chen, Haijian; Gimzewski, James K.; Reed, Jason; Abdel-Fattah, Tarek M.

2010-01-01

371

Application of SNPs in forensic casework: Identification of pathological and autoptical specimens due to sample mix-up  

Microsoft Academic Search

The analysis of single nucleotide polymorphisms (SNPs) together with conventional short tandem repeat (STR) and mitochondrial DNA (mtDNA) typing provide a forensic genetic approach for the identification of pathological and autoptical specimens in cases where the average length of DNA fragments is shorter than 150bp in highly degraded samples. We applied a forensic genetic approach to digesta accidentally left after

Yuzo Takada; Tomoharu Tokutomi; Jun Kanetake; Masahiro Mukaida

2009-01-01

372

Clostridium nitrophenolicum sp. nov., a novel anaerobic p-nitrophenol-degrading bacterium, isolated from a subsurface soil sample  

Microsoft Academic Search

was found to be capable of degrading p-nitrophenol (pNP) at a concentration of 0.5 mM under anaerobic conditions as revealed by HPLC analysis. The major fatty acids were C16:0 (28.02%), iso-C17:1 I\\/anteiso B (23.05%) and C14:0 (10.02%). The major polar lipid content was diphosphatidylglycerol. Strain 1DT showed highest 16S rRNA gene sequence similarity to Clostridium aciditolerans JW\\/YJL-B3T (98.2%) and similarity

K. Suresh; D. Prakash; N. Rastogi; R. K. Jain

2007-01-01

373

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

374

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

375

Fragmentation of Contaminant and Endogenous DNA in Ancient Samples Determined by Shotgun Sequencing; Prospects for Human Palaeogenomics  

PubMed Central

Background Despite the successful retrieval of genomes from past remains, the prospects for human palaeogenomics remain unclear because of the difficulty of distinguishing contaminant from endogenous DNA sequences. Previous sequence data generated on high-throughput sequencing platforms indicate that fragmentation of ancient DNA sequences is a characteristic trait primarily arising due to depurination processes that create abasic sites leading to DNA breaks. Methodology/Principals Findings To investigate whether this pattern is present in ancient remains from a temperate environment, we have 454-FLX pyrosequenced different samples dated between 5,500 and 49,000 years ago: a bone from an extinct goat (Myotragus balearicus) that was treated with a depurinating agent (bleach), an Iberian lynx bone not subjected to any treatment, a human Neolithic sample from Barcelona (Spain), and a Neandertal sample from the El Sidrón site (Asturias, Spain). The efficiency of retrieval of endogenous sequences is below 1% in all cases. We have used the non-human samples to identify human sequences (0.35 and 1.4%, respectively), that we positively know are contaminants. Conclusions We observed that bleach treatment appears to create a depurination-associated fragmentation pattern in resulting contaminant sequences that is indistinguishable from previously described endogenous sequences. Furthermore, the nucleotide composition pattern observed in 5? and 3? ends of contaminant sequences is much more complex than the flat pattern previously described in some Neandertal contaminants. Although much research on samples with known contaminant histories is needed, our results suggest that endogenous and contaminant sequences cannot be distinguished by the fragmentation pattern alone. PMID:21904610

Sanchez-Quinto, Federico; Ramirez, Oscar; Calafell, Francesc; Civit, Sergi; Lalueza-Fox, Carles

2011-01-01

376

DNA aggregation and cleavage in CGE induced by high electric field in aqueous solution accompanying electrokinetic sample injection.  

PubMed

The phenomenon of peak area decrease due to high injection voltage (Vinj , e.g. 10-30 kV, 200-600 V/cm in the 50 cm capillary) was found in the analysis of very dilute DNA fragments (<0.2 mg/L) by using high-sensitive electrokinetic supercharging-CGE. The possibility of DNA cleavage in aqueous solution was suggested, in addition to the aggregation phenomenon that is already known. The analysis of intentionally voltage-affected fragments (at 200 V/cm) also showed decreased peak areas depending on the time of the voltage being applied. Computer simulation suggested that a high electric field (a few kV/cm or more) could be generated partly between the electrode and the capillary end during electrokinetic injection (EKI) process. After thorough experimental verification, it was found that the factors affecting the damage during EKI were the magnitude of electric field, the distance between tips of electrode and capillary (De/c ), sample concentration and traveling time during EKI in sample vials. Furthermore, these factors are correlating with each other. A low conductivity of diluted sample would cause a high electric field (over a few hundred volts per centimeter), while the longer De/c results in a longer traveling time during EKI, which may cause a larger degree of damage (aggregation and cleavage) on the DNA fragments. As an important practical implication of this study, when the dilute DNA fragments (sub mg/L) are to be analyzed by CGE using EKI, injection voltage should be kept as low as possible. PMID:24242290

Ye, Xiaoxue; Mori, Satomi; Xu, Zhongqi; Hayakawa, Shinjiro; Hirokawa, Takeshi

2013-12-01

377

DNA Nanotechnology  

NSDL National Science Digital Library

In this activity, learners explore deoxyribonucleic acid (DNA), a nanoscale structure that occurs in nature. Learners extract a sample of DNA from split peas and put the sample in an Eppendorf tube to take home. Learners discover that nanoscientists study DNA to understand its biological function and use it to make other nanoscale materials and devices.

Nanoscale Informal Science Education Network

2014-06-10

378

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

379

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

380

Bioinformatics analysis of abnormal DNA methylation in muscle samples from monozygotic twins discordant for type 2 diabetes.  

PubMed

The present study aimed to examine the changes in DNA methylation of gene promoters associated with type 2 diabetes (T2D). The DNA methylation profile dataset GSE38291 was downloaded from the Gene Expression Omnibus database. A paired t?test was used to analyze differences in the DNA methylation of gene promoters between T2D and normal muscle samples. Gene Ontology (GO) enrichment analysis was performed using online tool, The Database for Annotation, Visualization and Integrated Discovery. Whole?Genome rVISTA was used to analyze the enriched transcription factor (TF) binding sites upstream of the transcription start site in the differentially methylated genes. A total of 38 genes, including Sirtuin 1, N?acetyltransferase 6, phospholipase A2 group XIIB and nuclear factor of activated T cells calcineurin?dependent 1, were identified to be differentially methylated between these two groups. One GO term, DNA geometric change (GO:0032392), was significantly enriched (P<0.05) by the hyper?methylated genes. In addition, the binding sites of one gene, zinc finger E?box binding homeobox 1, and three TFs, methyl CpG binding protein 2, TFEB and TFAP4, were significantly enriched in the hyper? and hypo?methylated genes, respectively. The resulting T2D?associated genes and potential TFs provided a novel insight into the molecular mechanisms underlying the pathology of T2D. These genes may become promising target genes for the development of treatments for T2D. PMID:25760736

Liu, Fei; Sun, Qianqian; Wang, Lingxiao; Nie, Shuangshuang; Li, Jun

2015-07-01

381

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

382

Use of a reamplification protocol improves sensitivity of detection of Mycobacterium tuberculosis in clinical samples by amplification of DNA.  

PubMed Central

We have compared the sensitivity and specificity of quantitative mycobacterial culture against results obtained by using the polymerase chain reaction for the detection of DNA from organisms of the Mycobacterium tuberculosis complex in 82 clinical specimens from patients suspected of having tuberculosis. Two amplification protocols were used, a standard amplification protocol, which amplifies a segment of the gene coding for the 65-kDa antigen, and a protocol in which the initial amplification products are reamplified with a second set of nested oligonucleotide primers. Although the standard amplification protocol gave positive results for 18 of 18 samples which grew greater than 100 CFU/ml and gave positive results in 4 of 35 specimens from patients with tuberculosis which were negative by culture, only 1 of 6 samples which grew less than 100 CFU/ml was positive. This lack of sensitivity could not be explained by the presence of inhibitors of Taq polymerase present in the original samples. In contrast, the reamplification protocol gave positive results for 24 of 24 samples which were positive by culture as well as for 13 of 35 samples from patients with tuberculosis which were negative by culture (overall sensitivity, 63%, P less than 0.02, compared with the standard amplification protocol and routine culture). Two of 23 samples from patients not diagnosed as having tuberculosis gave positive results when the standard amplification protocol was used, but no additional false-positive results were seen with the reamplification protocol (overall specificity, 91%). We conclude that the use of a reamplification protocol improves the sensitivity of detection of mycobacterial DNA in clinical samples without sacrificing specificity. The sensitivity of this approach appears to be superior to that of standard culture techniques. Images PMID:1909710

Pierre, C; Lecossier, D; Boussougant, Y; Bocart, D; Joly, V; Yeni, P; Hance, A J

1991-01-01

383

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. Rece