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1

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

PubMed

Extraction of DNA from forensic samples typically uses either an organic extraction protocol or solid phase extraction (SPE) and these methods generally involve numerous sample transfer, wash and centrifugation steps. Although SPE has been successfully adapted to the microdevice, it can be problematic because of lengthy load times and uneven packing of the solid phase. A closed-tube enzyme-based DNA preparation method has recently been developed which uses a neutral proteinase to lyse cells and degrade proteins and nucleases [14]. Following a 20 min incubation of the buccal or whole blood sample with this proteinase, DNA is polymerase chain reaction (PCR)-ready. This paper describes the optimization and quantitation of DNA yield using this method, and application to forensic biological samples, including UV- and heat-degraded whole blood samples on cotton or blue denim substrates. Results demonstrate that DNA yield can be increased from 1.42 (±0.21)ng/?L to 7.78 (±1.40)ng/?L by increasing the quantity of enzyme per reaction by 3-fold. Additionally, there is a linear relationship between the amount of starting cellular material added and the concentration of DNA in the solution, thereby allowing DNA yield estimations to be made. In addition, short tandem repeat (STR) profile results obtained using DNA prepared with the enzyme method were comparable to those obtained with a conventional SPE method, resulting in full STR profiles (16 of 16 loci) from liquid samples (buccal swab eluate and whole blood), dried buccal swabs and bloodstains and partial profiles from UV or heat-degraded bloodstains on cotton or blue denim substrates. Finally, the DNA preparation method is shown to be adaptable to glass or poly(methyl methacrylate) (PMMA) microdevices with little impact on STR peak height but providing a 20-fold reduction in incubation time (as little as 60 s), leading to a ?1 h reduction in DNA preparation time. PMID:22459950

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

2012-03-27

2

Modified midi- and mini-multiplex PCR systems for mitochondrial DNA control region sequence analysis in degraded samples.  

PubMed

Two multiplex polymerase chain reaction (PCR) systems (Midiplex and Miniplex) were developed for the amplification of the mitochondrial DNA (mtDNA) control region, and the efficiencies of the multiplexes for amplifying degraded DNA were validated using old skeletal remains. The Midiplex system consisted of two multiplex PCRs to amplify six overlapping amplicons ranging in length from 227 to 267 bp. The Miniplex system consisted of three multiplex PCRs to amplify 10 overlapping short amplicons ranging in length from 142 to 185 bp. Most mtDNA control region sequences of several 60-year-old and 400-500-year-old skeletal remains were successfully obtained using both PCR systems and consistent with those previously obtained by monoplex amplification. The multiplex system consisting of smaller amplicons is effective for mtDNA sequence analyses of ancient and forensic degraded samples, saving time, cost, and the amount of DNA sample consumed during analysis. PMID:23406419

Kim, Na Young; Lee, Hwan Young; Park, Sun Joo; Yang, Woo Ick; Shin, Kyoung-Jin

2013-02-13

3

Genetic identification of degraded DNA samples buried in different types of soil  

Microsoft Academic Search

Biological samples buried in different types of soil are often found in crime scenes. These samples are usually highly degraded which difficult their analysis. Several factors contribute to the degradation of biological material including temperature variation, humidity, UV light and especially the presence of microorganisms.Blood was collected from three non-related male donors and blood stains were made in fabrics such

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

2009-01-01

4

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

PubMed

Heterozygote imbalances leading to allele drop-outs and disproportionally large stutters leading to allele drop-ins are known stochastic phenomena related to STR typing of low-template DNA (LtDNA). The large stutters and the many drop-ins in typical STR stutter positions are artifacts from the PCR amplification of tandem repeats. These artifacts may be avoided by typing bi-allelic markers instead of STRs. In this work, the SNPforID multiplex assay was used to type LtDNA. A sensitized SNP typing protocol was introduced, that increased signal strengths without increasing noise and without affecting the heterozygote balance. Allele drop-ins were only observed in experiments with 25 pg of DNA and not in experiments with 50 and 100 pg of DNA. The allele drop-in rate in the 25 pg experiments was 0.06% or 100 times lower than what was previously reported for STR typing of LtDNA. A composite model and two different consensus models were used to interpret the SNP data. Correct profiles with 42-49 SNPs were generated from the 50 and 100 pg experiments, whereas a few incorrect genotypes were included in the generated profiles from the 25 pg experiments. With the strict consensus model, between 35 and 48 SNPs were correctly typed in the 25 pg experiments and only one allele drop-out (error rate: 0.07%) was observed in the consensus profiles. A total of 28 crime case samples were selected for typing with the sensitized SNPforID protocol. The samples were previously typed with old STR kits during the crime case investigation and only partial profiles (0-6 STRs) were obtained. Eleven of the samples could not be quantified with the Quantifiler™ Human DNA Quantification kit because of partial or complete inhibition of the PCR. For eight of these samples, SNP typing was only possible when the buffer and DNA polymerase used in the original protocol was replaced with the AmpF?STR(®) SEfiler Plus™ Master Mix, which was developed specifically for challenging forensic samples. All the crime case samples were successfully typed with the SNPforID multiplex assay and the match probabilities ranged from 1.1×10(-15) to 7.9×10(-23). In comparison, four of the samples could not be typed with the AmpF?STR(®) SEfiler Plus™ kit and the match probabilities were higher than 10(-7) for another six samples. PMID:23523365

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

2013-03-19

5

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

Microsoft Academic Search

A quadruplex real-time qPCR assay was developed to simultaneously assess total human DNA, human male DNA, DNA degradation and PCR inhibitors in forensic samples. Specifically, the assay utilizes a ?170–190bp target sequence that spans the TH01 STR locus to quantify total human DNA (nuTH01), a 137bp target sequence directly adjacent to the SRY gene to quantify human male DNA (nuSRY),

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

2008-01-01

6

Construction of two fluorescence-labeled non-combined DNA index system miniSTR multiplex systems to analyze degraded DNA samples in the Chinese Han Population.  

PubMed

MiniSTR loci have been demonstrated to be an effective approach in recovering genetic information from degraded specimens, because of the reduced PCR amplicon sizes which improved the PCR efficiency. Eight non-combined DNA index system miniSTR loci suitable for the Chinese Han Population were analyzed in 300 unrelated Chinese Han individuals using two novel five fluorescence-labeled miniSTR multiplex systems(multiplex I: D10S1248, D2S441, D1S1677 and D9S2157; multiplex II: D9S1122, D10S1435, D12ATA63, D2S1776 and Amelogenin). The allele frequency distribution and forensic parameters in the Chinese Han Population were reported in this article. The Exact Test demonstrated that all loci surveyed here were found to be no deviation from Hardy-Weinberg equilibrium. The accumulated power of discrimination and power of exclusion for the eight loci were 0.999999992 and 0.98, respectively. The highly degraded DNA from artificially degraded samples and the degraded forensic case work samples was assessed with the two miniSTR multiplex systems, and the results showed that the systems were quite effective. PMID:20715124

Bai, Xue; Li, Shujin; Cong, Bin; Li, Xia; Guo, Xia; He, Lujun; Ye, Jian; Pei, Li

2010-09-01

7

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

Microsoft Academic Search

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

Cordula Eichmann; Walther Parson

2008-01-01

8

DNA degradation with ozone.  

PubMed

DNA was ozonized in solution and the reaction was followed with polarimetry and with iodimetry. Polarimetry was used to determine the molar ratio DNA/O(3) when the DNA optical activity vanishes completely. At a molar ratio DNA/O(3)=2.3 the supramolecular structure of DNA collapses completely. Instead, iodimetry shows that the ozonolysis proceeds until all the nucleobases have been destroyed, an event which occurs at a molar ratio DNA/O(3)=1.1. The ozonolysis of DNA was also followed spectrophotometrically. DNA is reactive with ozone also in the solid state, as fixed bed. Clear indication about its oxidation derives from the FT-IR spectra from polarimetric measurements and from thermal analysis performed by thermogravimetric analysis (TGA), differential thermogravimetric analysis (DTG) and from differential thermal analysis (DTA). Particular remarkable is the fact that RNA has been found much less reactive toward ozone in the solid state than DNA. PMID:16616954

Cataldo, Franco

2006-04-17

9

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

PubMed

The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation. PMID:18369655

Eichmann, Cordula; Parson, Walther

2008-03-28

10

Persistent damage induces mitochondrial DNA degradation.  

PubMed

Considerable progress has been made recently toward understanding the processes of mitochondrial DNA (mtDNA) damage and repair. However, a paucity of information still exists regarding the physiological effects of persistent mtDNA damage. This is due, in part, to experimental difficulties associated with targeting mtDNA for damage, while sparing nuclear DNA. Here, we characterize two systems designed for targeted mtDNA damage based on the inducible (Tet-ON) mitochondrial expression of the bacterial enzyme, exonuclease III, and the human enzyme, uracil-N-glyosylase containing the Y147A mutation. In both systems, damage was accompanied by degradation of mtDNA, which was detectable by 6h after induction of mutant uracil-N-glycosylase and by 12h after induction of exoIII. Unexpectedly, increases in the steady-state levels of single-strand lesions, which led to degradation, were small in absolute terms indicating that both abasic sites and single-strand gaps may be poorly tolerated in mtDNA. mtDNA degradation was accompanied by the loss of expression of mtDNA-encoded COX2. After withdrawal of the inducer, recovery from mtDNA depletion occurred faster in the system expressing exonuclease III, but in both systems reduced mtDNA levels persisted longer than 144h after doxycycline withdrawal. mtDNA degradation was followed by reduction and loss of respiration, decreased membrane potential, reduced cell viability, reduced intrinsic reactive oxygen species production, slowed proliferation, and changes in mitochondrial morphology (fragmentation of the mitochondrial network, rounding and "foaming" of the mitochondria). The mutagenic effects of abasic sites in mtDNA were low, which indicates that damaged mtDNA molecules may be degraded if not rapidly repaired. This study establishes, for the first time, that mtDNA degradation can be a direct and immediate consequence of persistent mtDNA damage and that increased ROS production is not an invariant consequence of mtDNA damage. PMID:23721969

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

2013-05-27

11

STR analysis of artificially degraded DNA—results of a collaborative European exercise  

Microsoft Academic Search

Degradation of human DNA extracted from forensic stains is, in most cases, the result of a natural process due to the exposure of the stain samples to the environment. Experiences with degraded DNA from casework samples show that every sample may exhibit different properties in this respect, and that it is difficult to systematically assess the performance of routinely used

Peter M Schneider; Klaus Bender; Wolfgang R Mayr; Walther Parson; Bernadette Hoste; Ronny Decorte; Jan Cordonnier; Daniel Vanek; Niels Morling; Matti Karjalainen; C Marie-Paule Carlotti; Myriam Sabatier; Carsten Hohoff; Hermann Schmitter; Werner Pflug; Rainer Wenzel; Dieter Patzelt; Rüdiger Lessig; Peter Dobrowolski; Geraldine O’Donnell; Luciano Garafano; Marina Dobosz; Peter de Knijff; Bente Mevag; Ryszard Pawlowski; Leonor Gusmão; Maria Conceicao Vide; Antonio Alonso Alonso; Oscar Garc??a Fernández; Pilar Sanz Nicolás; Ann Kihlgreen; Walter Bär; Verena Meier; Anne Teyssier; Raphael Coquoz; Conxita Brandt; Ursula Germann; Peter Gill; Justine Hallett; Matthew Greenhalgh

2004-01-01

12

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

13

Authentication of forensic DNA samples  

Microsoft Academic Search

Over the past twenty years, DNA analysis has revolutionized forensic science, and has become a dominant tool in law enforcement. Today, DNA evidence is key to the conviction or exoneration of suspects of various types of crime, from theft to rape and murder. However, the disturbing possibility that DNA evidence can be faked has been overlooked. It turns out that

Dan Frumkin; Adam Wasserstrom; Ariane Davidson; Arnon Grafit

2010-01-01

14

DNA fingerprinting by sampled sequencing.  

PubMed Central

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

Brenner, S; Livak, K J

1989-01-01

15

Dual fluorescent labeling method to visualize plasmid DNA degradation.  

PubMed

The efficiency of nonviral vectors for gene delivery may be enhanced by understanding the key barriers that limit the translocation of the therapeutic DNA into the nucleus. One such barrier is the instability of DNA in the cytoplasm. In this work, we have developed a method to dual-label plasmid DNA to be utilized as a tool to elucidate DNA instability during its trafficking in the intracellular microenvironment. Plasmid DNA containing rhodamine and maleimide groups linked using peptide nucleic acid (PNA) linkers was utilized for conjugation. Covalent conjugation of the maleimide group with a second fluorophore, fluorescein, did not alter the electrophoretic mobility or the structural integrity of the DNA, as confirmed by gel electrophoresis. The intact DNA was visualized as a single color (yellow) due to the close proximity of the green and red fluorophores. DNA degradation was simulated using restriction endonucleases (BamH1 and PflMI) to cut the DNA at two or more sites resulting in color separation. Confocal time-lapse imaging was utilized to follow DNA stability upon incubation of Lipofectamine2000/dual-labeled DNA complexes in CHO-K1 cells. Yellow fluorescent voxels were seen in the cell cytoplasm indicating the presence of intact DNA. Red and green fluorescent voxels were also seen in a few cells, suggesting separation of the fluorophores and probable DNA degradation. The methodology developed in this report provides a new tracking tool for investigators to explore DNA degradation at the molecular level inside single cell. PMID:19086903

Srinivasan, Charudharshini; Siddiqui, Shafiuddin; Silbart, Lawrence K; Papadimitrakopoulos, Fotios; Burgess, Diane J

2009-01-01

16

Degradation and Transformability of DNA from Transgenic Leaves  

PubMed Central

The fate of transplastomic (chloroplast genome contains the transgene) tobacco plant DNA in planta was studied when the plant leaves were subjected to decay conditions simulating those encountered naturally, including grinding, incubation with cellulase or enzymes produced by Erwinia chrysanthemi, and attack by the plant pathogen Ralstonia solanacearum. Direct visualization of DNA on agarose gels, gene extraction yield (the number of amplifiable aadA sequences in extracted plant DNA), and the frequency that recipient bacteria can be transformed by plant DNA were used to evaluate the quality and quantity of plant DNA and the transgene. These measurements were used to monitor the physical and biological degradation of DNA inside decaying plant tissues. Our results indicate that while most of the DNA will be degraded inside plant cells, sufficient DNA persists to be released into the soil.

Ceccherini, MariaTeresa; Pote, John; Kay, Elisabeth; Van, Van Tran; Marechal, Joelle; Pietramellara, Giacomo; Nannipieri, Paolo; Vogel, Timothy M.; Simonet, Pascal

2003-01-01

17

A new SNP assay for identification of highly degraded human DNA.  

PubMed

There is growing evidence that the histone-DNA complexes found in nucleosomes offer protection from DNA degradation processes, including apoptotic events in addition to bacterial and environmental degradation. We sought to locate human nucleosome regions and build a catalogue of SNPs sited near the middle of these genomic segments that could be combined into a single PCR multiplex specifically for use with extremely degraded human genomic DNA samples. Using recently optimized bio-informatics tools for the reliable identification of nucleosome sites based on sequence motifs and their positions relative to known promoters, 1395 candidate loci were collected to construct an 18-plex single base extension assay. Genotyping performance of the nucleosome SNPs was tested using artificially degraded DNA and 24 casework samples where the likely state of degradation of DNA was established by comparison to profile completeness in four other forensic assays: a standard 15-plex STR identification test, a miniaturized STR multiplex and two autosomal SNP multiplexes. The nucleosome SNP assay gave genotyping success rates 6% higher than the best existing forensic SNP assay: the SNPforID Auto-2 29-plex and significantly higher than the mini-STR assay. The nucleosome SNPs we located and combined therefore provide a new type of marker set that can be used to supplement existing approaches when the analysed DNA is likely to be extremely degraded and may fail to give sufficient STR genotypes for a reliable identification. PMID:21908243

Freire-Aradas, A; Fondevila, M; Kriegel, A-K; Phillips, C; Gill, P; Prieto, L; Schneider, P M; Carracedo, A; Lareu, M V

2011-09-09

18

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

PubMed

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

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

2011-01-26

19

Oxidative stress induces degradation of mitochondrial DNA  

Microsoft Academic Search

Mitochondrial DNA (mtDNA) is located in close prox- imity of the respiratory chains, which are the main cellular source of reactive oxygen species (ROS). ROS can induce oxidative base lesions in mtDNA and are believed to be an important cause of the mtDNA mutations, which accumulate with aging and in diseased states. However, recent studies indicate that cumulative levels of

Inna Shokolenko; Natalia Venediktova; Alexandra Bochkareva; Glenn L. Wilson; Mikhail F. Alexeyev

2009-01-01

20

Exonuclease Degradation of DNA Studied by Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

Here we developed an accurate method for kinetic analysis of enzymatic degradation processes of double and\\/or single-stranded DNA\\/oligonucleotides using fluorescent reporter dyes. 217-bp DNA fragments were produced by polymerase chain reaction and cleaved by the 3? to 5? exonuclease activity of T7-DNA polymerase. The analysis of the products was performed by Fluorescence Correlation Spectroscopy measuring autocorrelation amplitudes and diffusion times.

Zeno Földes-Papp; Per Thyberg; Sofie Björling; Arne Holmgen; Rudolf Rigler

1997-01-01

21

Anaerobic methyl tert-butyl ether-degrading microorganisms identified in wastewater treatment plant samples by stable isotope probing.  

PubMed

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; Cupples, Alison M

2012-02-10

22

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

PubMed Central

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

Sun, Weimin; Sun, Xiaoxu

2012-01-01

23

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

NASA Astrophysics Data System (ADS)

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

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

2005-05-01

24

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

25

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

26

Direct uptake and degradation of DNA by lysosomes.  

PubMed

Lysosomes contain various hydrolases that can degrade proteins, lipids, nucleic acids and carbohydrates. We recently discovered "RNautophagy," an autophagic pathway in which RNA is directly taken up by lysosomes and degraded. A lysosomal membrane protein, LAMP2C, a splice variant of LAMP2, binds to RNA and acts as a receptor for this pathway. In the present study, we show that DNA is also directly taken up by lysosomes and degraded. Like RNautophagy, this autophagic pathway, which we term "DNautophagy," is dependent on ATP. The cytosolic sequence of LAMP2C also directly interacts with DNA, and LAMP2C functions as a receptor for DNautophagy, in addition to RNautophagy. Similarly to RNA, DNA binds to the cytosolic sequences of fly and nematode LAMP orthologs. Together with the findings of our previous study, our present findings suggest that RNautophagy and DNautophagy are evolutionarily conserved systems in Metazoa. PMID:23839276

Fujiwara, Yuuki; Kikuchi, Hisae; Aizawa, Shu; Furuta, Akiko; Hatanaka, Yusuke; Konya, Chiho; Uchida, Kenko; Wada, Keiji; Kabuta, Tomohiro

2013-05-21

27

Direct uptake and degradation of DNA by lysosomes  

PubMed Central

Lysosomes contain various hydrolases that can degrade proteins, lipids, nucleic acids and carbohydrates. We recently discovered “RNautophagy,” an autophagic pathway in which RNA is directly taken up by lysosomes and degraded. A lysosomal membrane protein, LAMP2C, a splice variant of LAMP2, binds to RNA and acts as a receptor for this pathway. In the present study, we show that DNA is also directly taken up by lysosomes and degraded. Like RNautophagy, this autophagic pathway, which we term “DNautophagy,” is dependent on ATP. The cytosolic sequence of LAMP2C also directly interacts with DNA, and LAMP2C functions as a receptor for DNautophagy, in addition to RNautophagy. Similarly to RNA, DNA binds to the cytosolic sequences of fly and nematode LAMP orthologs. Together with the findings of our previous study, our present findings suggest that RNautophagy and DNautophagy are evolutionarily conserved systems in Metazoa.

Fujiwara, Yuuki; Kikuchi, Hisae; Aizawa, Shu; Furuta, Akiko; Hatanaka, Yusuke; Konya, Chiho; Uchida, Kenko; Wada, Keiji; Kabuta, Tomohiro

2013-01-01

28

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

Microsoft Academic Search

The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined. The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained. The colony hybridization technique allowed detection of

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

1985-01-01

29

Improved analysis of long STR amplicons from degraded single source and mixed DNA.  

PubMed

DNA profiles from degraded samples often suffer from information loss at the longer short tandem repeat (STR) loci. Sensitising the reactions, either by performing additional PCR cycles or increasing the capillary electrophoresis injection settings, carries the risk of over-amplifying or overloading the shorter fragments. We explored whether profiling of degraded DNA can be improved by preferential capturing of the longer amplified fragments. To this aim, a post-PCR purification protocol was developed that is based on AMPure XP beads that have size-selective properties. A comparison was made with an unselective post-PCR purification system (DTR gel filtration) and no purification of the PCR products. Besides a set of differently and serially degraded single source samples, unequal mixtures of degraded DNAs were analysed, in order to extract more genotyping information for the minor contributor without overloading the major component at the shorter amplicons. Purification by the AMPure protocol resulted in higher peak heights especially for the longer amplicons, while DTR gel filtration gave higher peaks for all amplicon sizes. Both purification methods presented more detected alleles, with the AMPure protocol performing slightly better, on average. In conclusion, the in-house developed AMPure protocol can be employed to improve STR profile analysis of degraded single source and (unequally) mixed DNA samples. PMID:23306520

Westen, Antoinette A; van der Gaag, Kristiaan J; de Knijff, Peter; Sijen, Titia

2013-01-10

30

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2010 CFR

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

2009-07-01

31

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

32

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

33

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2010 CFR

...2007-07-01 2007-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing §...

2007-07-01

34

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2010 CFR

...2008-07-01 2008-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing §...

2008-07-01

35

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2010 CFR

...2006-07-01 2006-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing §...

2006-07-01

36

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2010 CFR

...2001-07-01 2001-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing §...

2001-07-01

37

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2010 CFR

...2005-07-01 2005-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing §...

2005-07-01

38

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2010 CFR

...2004-07-01 2004-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing §...

2004-07-01

39

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2010 CFR

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

2002-07-01

40

28 CFR 28.12 - Collection of DNA samples.  

Code of Federal Regulations, 2010 CFR

...2003-07-01 2003-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing §...

2003-07-01

41

Prevention of oxidative DNA degradation by copper-binding peptides.  

PubMed

Free divalent ions of copper (Cu) are capable of generating radical species such as hydroxyl radicals in the presence of hydrogen peroxide or ascorbic acid through Harbor-Weiss-like reactions under physiological conditions. It has been reported that radical-mediated damage to DNA molecules in animal cells leads to programmed cell death. Hence it is important to seek for methods to prevent Cu-mediated DNA damage. In this study we identified on effect of Cu binding of short peptides (chiefly Gly-Gly-His tripeptide) in the prevention of DNA degradation caused by Cu-mediated reactions in the presence of hydrogen peroxide and of ascorbic acid. PMID:21737913

Yokawa, Ken; Kagenishi, Tomoko; Kawano, Tomonori

2011-07-07

42

Characterization of new miniSTR loci to aid analysis of degraded DNA.  

PubMed

A number of studies have demonstrated that successful analysis of degraded DNA specimens from mass disasters or forensic evidence 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 125 bp 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, and D22S1045, arranged into two miniSTR triplexes. All loci show a moderate degree of polymorphism among 474 U.S. population samples tested and were reliable and sensitive to at least 100 pg of DNA template under controlled laboratory conditions and pristine DNA samples. The utility of these new loci were confirmed in comparing the success of the miniSTR assays for typing degraded bone samples while partial profiles were observed with the majority of the samples using a commercial STR kit. PMID:15830996

Coble, Michael D; Butler, John M

2005-01-01

43

Degradation of polycyclic aromatic hydrocarbons during simulated stack gas sampling  

SciTech Connect

The formation of sampling artifacts due to exposure to NO, NO/sub 2/, SO/sub 2/, and SO/sub 3/ has been studied. The sampling method was tested in a thermostatic oven attached to a smoke generator and involves filtration of particles at stack gas temperature, condensation, and adsorption on Amberlite XAD-2. A substantial degradation of reactive PAH took place in all steps during sampling. The reactivity increased in the presence of acid. The reaction products formed included 9-nitroanthracene, 1-nitropyrene, 10-nitrobenz(a)anthracene, nitrobenzo(a)pyrene, and several polycyclic ketones, aldehydes, and guinones, but the main reaction products have not been identified so far.

Brorstroem-Lunden, E.; Lindskog, A.

1985-01-01

44

Salttolerant phenol-degrading microorganisms isolated from Amazonian soil samples  

Microsoft Academic Search

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 faecalis 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

Artur Eduardo Ribeiro Bastos; David Henry Moon; Antonio Rossi; Jack Thomas Trevors; Siu Mui Tsai

2000-01-01

45

Interpreting anonymous DNA samples from mass disasters - probabilistic forensic inference using genetic markers  

Microsoft Academic Search

Motivation: The problem of identifying victims in a mass disa- ster using DNA fingerprints involves a scale of computation that requires efficient and accurate algorithms. In a typical scenario there are hundreds of samples taken from remains that must be matched to the pedigrees of the alleged victim's surviving relatives. Moreover the samples are often degraded due to heat and

Tien-ho Lin; Eugene W. Myers; Eric P. Xing

2006-01-01

46

Identification of hydrocarbon-degrading bacteria in soil by reverse sample genome probing.  

PubMed

Bacteria with limited genomic cross-hybridization were isolated from soil contaminated with C5+, a mixture of hydrocarbons, and identified by partial 16S rRNA sequencing. Filters containing denatured genomic DNAs were used in a reverse sample genome probe (RSGP) procedure for analysis of the effect of an easily degradable compound (toluene) and a highly recalcitrant compound (dicyclopentadiene [DCPD]) on community composition. Hybridization with labeled total-community DNA isolated from soil exposed to toluene indicated enrichment of several Pseudomonas spp., which were subsequently found to be capable of toluene mineralization. Hybridization with labeled total-community DNA isolated from soil exposed to DCPD indicated enrichment of a Pseudomonas sp. or a Sphingomonas sp. These two bacteria appeared capable of producing oxygenated DCPD derivatives in the soil environment, but mineralization could not be shown. These results demonstrate that bacteria, which metabolize degradable or recalcitrant hydrocarbons, can be identified by the RSGP procedure. PMID:16349504

Shen, Y; Stehmeier, L G; Voordouw, G

1998-02-01

47

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

PubMed Central

Bacteria with limited genomic cross-hybridization were isolated from soil contaminated with C5+, a mixture of hydrocarbons, and identified by partial 16S rRNA sequencing. Filters containing denatured genomic DNAs were used in a reverse sample genome probe (RSGP) procedure for analysis of the effect of an easily degradable compound (toluene) and a highly recalcitrant compound (dicyclopentadiene [DCPD]) on community composition. Hybridization with labeled total-community DNA isolated from soil exposed to toluene indicated enrichment of several Pseudomonas spp., which were subsequently found to be capable of toluene mineralization. Hybridization with labeled total-community DNA isolated from soil exposed to DCPD indicated enrichment of a Pseudomonas sp. or a Sphingomonas sp. These two bacteria appeared capable of producing oxygenated DCPD derivatives in the soil environment, but mineralization could not be shown. These results demonstrate that bacteria, which metabolize degradable or recalcitrant hydrocarbons, can be identified by the RSGP procedure.

Shen, Yin; Stehmeier, Lester G.; Voordouw, Gerrit

1998-01-01

48

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

NASA Astrophysics Data System (ADS)

This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the occurrence of a red fluorescence signal due to fluorescence resonance energy transfer (FRET). DNase in biological samples was detected using DFProbes and the fluorescence imaging in living cells was performed using DFprobe-modified Au nanoparticles. The results show that DFProbes have good responses to DNase, and can clearly visualize the degradation of exogenous DNA in cells in real time. The well-designed probes might be useful in tracing the dynamic changes of exogenous DNA and nanocarriers in vitro and in vivo.This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the occurrence of a red fluorescence signal due to fluorescence resonance energy transfer (FRET). DNase in biological samples was detected using DFProbes and the fluorescence imaging in living cells was performed using DFprobe-modified Au nanoparticles. The results show that DFProbes have good responses to DNase, and can clearly visualize the degradation of exogenous DNA in cells in real time. The well-designed probes might be useful in tracing the dynamic changes of exogenous DNA and nanocarriers in vitro and in vivo. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr12005d

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

2012-03-01

49

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

50

Automatable full demineralization DNA extraction procedure from degraded skeletal remains.  

PubMed

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

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

2011-08-31

51

The application of magnetic bead hybridization for the recovery and STR amplification of degraded and inhibited forensic DNA.  

PubMed

A common problem in the analysis of forensic DNA evidence is the presence of environmentally degraded and inhibited DNA. Such samples produce a variety of interpretational problems such as allele imbalance, allele dropout and sequence specific inhibition. In an attempt to develop methods to enhance the recovery of this type of evidence, magnetic bead hybridization has been applied to extract and preconcentrate DNA sequences containing short tandem repeat (STR) alleles of interest. In this work, genomic DNA was fragmented by heating, and sequences associated with STR alleles were selectively hybridized to allele-specific biotinylated probes. Each particular biotinylated probe-DNA complex was bound to streptavidin-coated magnetic beads using enabling enrichment of target DNA sequences. Experiments conducted using degraded DNA samples, as well as samples containing a large concentration of inhibitory substances, showed good specificity and recovery of missing alleles. Based on the favorable results obtained with these specific probes, this method should prove useful as a tool to improve the recovery of alleles from degraded and inhibited DNA samples. PMID:21706494

Wang, Jing; McCord, Bruce

2011-06-01

52

Forensic Mitochondrial DNA Analysis of 116 Casework Skeletal Samples  

Microsoft Academic Search

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

Kimberlyn Nelson; Terry Melton

2007-01-01

53

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

PubMed

Sex determination is of paramount importance in forensic anthropology. Numerous anthropological methods have been described, including visual assessments and various measurements of bones. Nevertheless, whatever the method used, the percentage of correct classification of a single bone usually varies between 80% and 95%, due to significant intra- and inter-population variations, and sometimes variations coming from secular trends. DNA is increasingly used in a forensic context. But forensic DNA extraction from bone raises several issues, because the samples are very often badly altered and/or in very small quantity. Nuclear DNA is difficult to get from degraded samples, according to low copy number, at least in comparison with mitochondrial DNA. In a forensic context (as in a paeleoanthropological context) DNA sex determination is usually complicated by the weak amount of DNA, the degraded nature of nucleic acids, the presence of enzymatic inhibitors in DNA extracts, the possible faint amplification of Y band and the risk of contamination during either excavation or manipulation of samples. The aim of this work was to compare three methods of DNA sex determination from bones: procedure #1 using a single PCR amplification, procedure #2 using a double PCR amplification, and procedure #3 adding bleaching for decontamination of the bone, instead of simply rubbing the bone. These processes were applied to samples of bones (49 samples coming from 39 individuals) that were in various states of post mortem alteration. The main results are the following. (i) No DNA could be extracted from three skulls (parietal bones, mastoid process), the compact bone of one rib, and the diaphysis of one femur; (ii) there was a contamination in three skulls; and (iii) the Y band did not appear in two male cases, with one of the three procedures (male tibia, procedure #2) and with procedures #2 and #3 (male femur). This study emphasises the main issue while working with altered bones: the impossibility to extract DNA in some cases, and, worth of all, the contamination of the sample or the faint amplification of Y band which leads to a wrong sex answer. Multiple and significant precautions have to be taken to avoid such difficulties. PMID:23937932

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

2013-05-01

54

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

PubMed Central

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

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

2012-01-01

55

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

PubMed

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

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

2012-08-29

56

Singlet oxygen mediated DNA degradation by copper nanoparticles: potential towards cytotoxic effect on cancer cells  

Microsoft Academic Search

The DNA degradation potential and anti-cancer activities of copper nanoparticles of 4-5 nm size are reported. A dose dependent\\u000a degradation of isolated DNA molecules by copper nanoparticles through generation of singlet oxygen was observed. Singlet oxygen\\u000a scavengers such as sodium azide and Tris [hydroxyl methyl] amino methane were able to prevent the DNA degradation action of\\u000a copper nanoparticles confirming the

Gregor P Jose; Subhankar Santra; Swadhin K Mandal; Tapas K Sengupta

2011-01-01

57

Interpretation of the Apollo 14 Thermal Degradation Sample experiment  

NASA Astrophysics Data System (ADS)

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

Gaier, James R.

2012-09-01

58

Automated DNA extraction for large numbers of plant samples.  

PubMed

The method described here is a rapid, total DNA extraction procedure applicable to a large number of plant samples requiring pathogen detection. The procedure combines a simple and quick homogenization step of crude extracts with DNA extraction based upon the binding of DNA to magnetic beads. DNA is purified in an automated process in which the magnetic beads are transferred through a series of washing buffers. The eluted DNA is suitable for efficient amplification in PCR reactions. PMID:22987412

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

2013-01-01

59

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

Microsoft Academic Search

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

Nicolas Gryson

2010-01-01

60

Human papillomavirus DNA in urine samples compared with that in simultaneously collected urethra and cervix samples.  

PubMed Central

A polymerase chain reaction was used to evaluate the occurrence of human papillomavirus (HPV) DNA in urine samples compared with that in urethra and cervix samples simultaneously collected with brushes. Of 138 presumably healthy military conscripts, 12 (8%) had HPV DNA-positive urethra samples and 8 (5%) had HPV DNA-positive urine samples. Both the urine and urethra cell samples of five men were positive, with identical types found in the paired specimens. Seven had HPV DNA-positive urethra samples only, and three had HPV DNA-positive urine samples only. Five of 7 urethra samples from males and 11 of 12 urethra samples from females, who were among patients consulting a clinic for adolescents, were positive for HPV DNA. Among those patients whose urethras were positive for HPV DNA, the corresponding urine samples of 3 of the 5 men and all the 11 women were also positive, with one or two HPV types being in common within the paired samples. Among female patients referred to a colposcopy clinic, 49% (241 of 489) of the cervical cell samples and 38% (187 of 489) of the urine specimens were found to be HPV DNA positive. Of the patients whose cervixes were positive for HPV DNA, 65% (158 of 241) of the simultaneously collected urine samples were also positive for HPV DNA. On the other hand, 84% (158 of 187) of the patients with HPV DNA in their urine also had HPV DNA in their cervical samples. Although not all individuals with genital HPV infections could be identified as HPV positive by analysis of urine samples, at least in epidemiological surveys in which invasive samples are difficult to obtain, such as from children, analysis of urine could be an alternative means of identifying HPV DNA.

Forslund, O; Hansson, B G; Rymark, P; Bjerre, B

1993-01-01

61

Stability of linear DNA in recA mutant Escherichia coli cells reflects ongoing chromosomal DNA degradation.  

PubMed Central

To study the fate of linear DNA in Escherichia coli cells, we linearized plasmid DNA at a specific site in vivo and monitored its behavior in recA mutant cells deficient in recombinational repair. Earlier, we had found that in wild-type (WT) cells linearized DNA is degraded to completion by RecBCD nuclease. We had also found that in WT cells chi sites on linear DNA inhibit RecBCD degradation by turning off its nucleolytic activities. Now we report that chi sites do not work in the absence of the RecA protein, suggesting that RecA is required in vivo to turn off the degradative activities of the RecBCD enzyme. We also report that the degradation of linearized plasmid DNA, even devoid of chi sites, is never complete in recA cells. Investigation of this linear DNA stability indicates that a fraction of recA cells are recBC phenocopies due to ongoing chromosomal DNA degradation, which titrates RecBCD nuclease. A possible role for RecBCD-promoted DNA degradation in controlling chromosomal DNA replication in E. coli is discussed.

Kuzminov, A; Stahl, F W

1997-01-01

62

Electrochemical DNA biosensor for analysis of wastewater samples  

Microsoft Academic Search

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

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

2002-01-01

63

Rapid cleanup of bacterial DNA from samples containing aerosol contaminants  

NASA Astrophysics Data System (ADS)

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

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

1999-01-01

64

DNA identification of Salvia divinorum samples.  

PubMed

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

Murphy, Terence M; Bola, Gurpreet

2012-05-09

65

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

PubMed

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

2012-11-07

66

An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.  

PubMed

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications. PMID:16766515

Lee, Cheryl I P; Leong, Siew Hong; Png, Adrian E H; Choo, Keng Wah; Syn, Christopher; Lim, Dennis T H; Law, Hai Yang; Kon, Oi Lian

2006-03-27

67

Getting DNA copy numbers without control samples  

PubMed Central

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

2012-01-01

68

Preparation of DNA and nucleoprotein samples for AFM imaging  

Microsoft Academic Search

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

Yuri L. Lyubchenko

2011-01-01

69

Improved Detection of Male DNA in Post-Coital Samples.  

National Technical Information Service (NTIS)

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

A. van Daal H. Lubenow J. Ballantyne

2012-01-01

70

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

PubMed

Ecological and conservation genetics require sampling of organisms in the wild. Appropriate preservation of the collected samples, usually by cryostorage, is key to the quality of the genetic data obtained. Nevertheless, cryopreservation in the field to ensure RNA and DNA stability is not always possible. We compared several nucleic acid preservation solutions appropriate for field sampling and tested them on rat (Rattus rattus) blood, ear and tail tip, liver, brain and muscle. We compared the efficacy of a nucleic acid preservation (NAP) buffer for DNA preservation against 95% ethanol and Longmire buffer, and for RNA preservation against RNAlater (Qiagen) and Longmire buffer, under simulated field conditions. For DNA, the NAP buffer was slightly better than cryopreservation or 95% ethanol, but high molecular weight DNA was preserved in all conditions. The NAP buffer preserved RNA as well as RNAlater. Liver yielded the best RNA and DNA quantity and quality; thus, liver should be the tissue preferentially collected from euthanized animals. We also show that DNA persists in nonpreserved muscle tissue for at least 1 week at ambient temperature, although degradation is noticeable in a matter of hours. When cryopreservation is not possible, the NAP buffer is an economical alternative for RNA preservation at ambient temperature for at least 2 months and DNA preservation for at least 10 months. PMID:23617785

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

2013-04-26

71

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

2009-12-15

72

Cell-free plasma DNA and purine nucleotide degradation markers following weightlifting exercise.  

PubMed

The present study aimed to investigate the acute effects of a single bout of high-intensive strength training on the production of cell-free plasma DNA (cf-DNA), as well as on the degradation of purine nucleotides as assessed by the concentration of xanthine (XA) and hypoxanthine (HX) in urine and serum. Twelve trained weightlifters performed six sets of six lifting exercises with 90-95% of the one repetition maximum. Blood samples and urine were obtained 1 h before training, immediately after finishing the exercise session and following 2 h of recovery. Cf-DNA, HX, and XA (in serum) significantly increased (P < 0.05-P < 0.001) immediately after heavy lifting exercise when compared with baseline levels, and significantly decreased (P < 0.05-P < 0.001) after 2 h of recovery. These results indicate that, cf-DNA and oxypurines might be relevant biomarkers for cellular damage, mechanical, energetic, and/or ischemic stress in context with exercise. PMID:20577758

Atamaniuk, Johanna; Vidotto, Claudia; Kinzlbauer, Markus; Bachl, Norbert; Tiran, Beate; Tschan, Harald

2010-06-26

73

Preparation of DNA and nucleoprotein samples for AFM imaging  

PubMed Central

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

Lyubchenko, Yuri L.

2010-01-01

74

Preparation of DNA and nucleoprotein samples for AFM imaging.  

PubMed

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

2010-09-09

75

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

76

XPB Mediated Retroviral cDNA Degradation Coincides with Entry to the Nucleus  

PubMed Central

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 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.; Roddick, William; Hoellerbauer, Pia; Fishel, Richard

2010-01-01

77

Transcription-Dependent Degradation of Topoisomerase I-DNA Covalent Complexes  

PubMed Central

Topoisomerase I (Top I)-DNA covalent complexes represent a unique type of DNA lesion whose repair and processing remain unclear. In this study, we show that Top I-DNA covalent complexes transiently arrest RNA transcription in normal nontransformed cells. Arrest of RNA transcription is coupled to activation of proteasomal degradation of Top I and the large subunit of RNA polymerase II. Recovery of transcription occurs gradually and depends on both proteasomal degradation of Top I and functional transcription-coupled repair (TCR). These results suggest that arrest of the RNA polymerase elongation complex by the Top I-DNA covalent complex triggers a 26S proteasome-mediated signaling pathway(s) leading to degradation of both Top I and the large subunit of RNA polymerase II. We propose that proteasomal degradation of Top I and RNA polymerase II precedes repair of the exposed single-strand breaks by TCR.

Desai, Shyamal D.; Zhang, Hui; Rodriguez-Bauman, Alexandra; Yang, Jin-Ming; Wu, Xiaohua; Gounder, Murugesan K.; Rubin, Eric H.; Liu, Leroy F.

2003-01-01

78

Forensic DNA Sampling and the England and Wales National DNA Database: A Sceptical Approach  

Microsoft Academic Search

This paper explores possible implications of the rapid expansion of the England and Wales National DNA Database (NDNAD), and\\u000a the current DNA sampling of offenders and the retention of samples. A precis of the justifications enunciated for the NDNAD\\u000a is followed by a sceptic's rebuttal and wider analysis of the impact of the growth of forensic DNA testing. It is

Carole Mccartney

2004-01-01

79

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

PubMed

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

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

2004-04-01

80

Bioaugmentation of Bromoamine Acid Degradation with Sphingomonas xenophaga QYY and DNA Fingerprint Analysis of Augmented Systems  

Microsoft Academic Search

One high-effective bromoamine acid (1-amino-4-bromoanthraquinone-2-sulfonic acid, BAA) degrading strain was isolated previously\\u000a with the ability to use BAA as sole source of carbon and nitrogen. It was identified as Sphingomonas xenophaga QYY by 16S rDNA sequence analysis and physio-biochemical tests. In this study, bioaugmentation of BAA degradation with suspended\\u000a and immobilized cells of strain QYY was investigated. The optimal degradation

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

2006-01-01

81

The protein degradation response of Saccharomyces cerevisiae to classical DNA-damaging agents.  

PubMed

Genome wide experiments indicate both proteasome- and vacuole-mediated protein degradation modulate sensitivity to classical DNA-damaging agents. Here, we show that global protein degradation is significantly increased upon methyl methanesulfonate (MMS) exposure. In addition, global protein degradation is similarly increased upon exposure to 4-nitroquinoline-N-oxide (4NQO) and UV and, to a lesser extent, tert-butyl hydroperoxide. The proteasomal inhibitor MG132 decreases both MMS-induced and 4NQO-induced protein degradation, while addition of the vacuolar inhibitor phenylmethanesulfonyl fluoride does not. The addition of both inhibitors grossly inhibits cell growth upon MMS exposure over and above the growth inhibition induced by MMS alone. The MMS-induced protein degradation response remains unchanged in several ubiquitin-proteasome and vacuolar mutants, presumably because these mutants are not totally deficient in either essential pathway. Furthermore, MMS-induced protein degradation is independent of Mec1, Mag1, Rad23, and Rad6, suggesting that the protein degradation response is not transduced through the classical Mec1 DNA damage response pathway or through repair intermediates generated by the base excision, nucleotide excision, or postreplication-DNA repair pathways. These results identify the regulation of protein degradation as an important factor in the recovery of cells from toxicity induced by classical DNA-damaging agents. PMID:18020423

Burgis, Nicholas E; Samson, Leona D

2007-11-20

82

A DNA methylation fingerprint of 1628 human samples  

PubMed Central

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

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

2012-01-01

83

Interactions of DNA with clay minerals and soil colloidal particles and protection against degradation by DNase.  

PubMed

Adsorption, desorption, and degradation by nucleases of DNA on four different colloidal fractions from a Brown soil and clay minerals were studied. The adsorption of DNase I and the structures of native DNA, adsorbed and desorbed, were also investigated by Fourier Transform Infrared (FTIR), circular dichroism (CD), and fluorescence spectroscopy, to determine the protection mechanism of DNA molecules by soil colloids and minerals against enzymatic degradation. Kaolinite exhibited the highest adsorption affinity for DNA among the examined soil colloids and clay minerals. In comparison with organomineral complexes (organic clays), DNA was tightly adsorbed by H2O2-treated clays (inorganic clays). FTIR spectra showed that the binding of DNA on kaolinite and inorganic clays changed its conformation from the B-form to the Z-form, whereas montmorillonite and organic clays retained the original B-form of DNA. A structural change from the B- to the C-form in DNA molecules desorbed from kaolinite was observed by CD spectroscopy and confirmed by fluorescence spectroscopy. The presence of soil colloids and minerals provided protection to DNA against degradation by DNase I. The higher level of protection was found with montmorillonite and organic clays compared to kaolinite and inorganic clays. The protection of DNA against nuclease degradation by soil colloids and minerals is apparently not controlled by the adsorption affinity of DNA molecules for the colloids and the conformational change of bound DNA. The higher stability of DNA seemed to be attributed mainly to the presence of organic matter in the system and the adsorption of nucleases on soil colloids and minerals. The information obtained in this study is of fundamental significance for the understanding of the behavior of extracellular DNA in soil environment. PMID:16719099

Cai, Peng; Huang, Qiao-Yun; Zhang, Xue-Wen

2006-05-01

84

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

PubMed

The innate immune system is important for control of infections, including herpesvirus infections. Intracellular DNA potently stimulates antiviral IFN responses. It is known that plasmacytoid dendritic cells sense herpesvirus DNA in endosomes via TLR9 and that nonimmune tissue cells can sense herpesvirus DNA in the nucleus. However, it remains unknown how and where myeloid cells, such as macrophages and conventional dendritic cells, detect infections with herpesviruses. In this study, we demonstrate that the HSV-1 capsid was ubiquitinated in the cytosol and degraded by the proteasome, hence releasing genomic DNA into the cytoplasm for detection by DNA sensors. In this context, the DNA sensor IFN-?-inducible 16 is important for induction of IFN-? in human macrophages postinfection with HSV-1 and CMV. Viral DNA localized to the same cytoplasmic regions as did IFN-?-inducible 16, with DNA sensing being independent of viral nuclear entry. Thus, proteasomal degradation of herpesvirus capsids releases DNA to the cytoplasm for recognition by DNA sensors. PMID:23345332

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

2013-01-23

85

Safeguarding forensic DNA reference samples with nullomer barcodes.  

PubMed

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

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

2013-04-17

86

Summary Statistics of Neutral Mutations in Longitudinal DNA Samples  

PubMed Central

Longitudinal samples of DNA sequences are the DNA sequences sampled from the same population at different time points. For fast evolving organisms, e.g. RNA virus, these kind of samples have increasingly been used to study the evolutionary process in action. Longitudinal samples provide some interesting new summary statistics of genetic variation, such as the frequency of mutation of size i in one sample and size j in another, the average number of mutations accumulated since the common ancestor of two sequences each from a different sample, and number of private, shared and fixed mutations within samples. To make the results more applicable, we used in this study a general two-sample model, which assumes two longitudinal samples were taken from the same measurably evolving population. Inspired by the HIV study, we also studied a two-sample-two-stage model, which is a special case of two-sample model and assumes a treatment after the first sampling instantaneously changes the population size. We derived the formulas for calculating statistical properties, e.g. expectations, variances and covariances, of these new summary statistics under the two models. Potential applications of these results were discussed.

Liu, Xiaoming; Fu, Yun-Xin

2011-01-01

87

DNA degradation in chilled fresh chicken studied with the neutral comet assay  

Microsoft Academic Search

DNA degradation in fresh chicken was studied with the neutral comet assay. Chicken legs were stored in a refrigerator at\\u000a 2–4??°C. DNA from muscle tissue was analysed independently by the authors in Mol and Uppsala after 1–12 days. The cells were\\u000a imbedded in agarose on a microscope slide, lysed and subjected to electrophoresis causing DNA and its fragments to migrate

H. Cerda; G. Koppen

1998-01-01

88

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

PubMed Central

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

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

2012-01-01

89

Preamplification Procedure for the Analysis of Ancient DNA Samples  

PubMed Central

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

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

2013-01-01

90

Defective degradation of bacterial DNA by phagocytes from patients with systemic and discoid lupus erythematosus.  

PubMed Central

The digestion of bacterial DNA by peripheral blood monocytes was impaired both in patients with systemic lupus erythematosus (SLE) and discoid lupus erythematosus (DLE). The monocytes of these patients had both a small quantitative defect in the solubilization of DNA and a marked qualitative defect in the extent to which this DNA was degraded. In addition, neutrophils from patients with SLE released significantly less high molecular-weight DNA than control cells. Digestion of bacterial RNA and protein by phagocytes was not defective in either disease. The reduced digestion of DNA by phagocytes resulted in concomitantly larger amounts of high molecular-weight DNA remaining in these cells. Such sequestration of DNA may contribute to the persistence of fairly large DNA fragments in the tissue of patients with lupus erythematosus.

Roberts, P J; Isenberg, D A; Segal, A W

1987-01-01

91

A new multiplex-PCR comprising autosomal and y-specific STRs and mitochondrial DNA to analyze highly degraded material.  

PubMed

The analysis of short tandem repeats is one of the most powerful tools in forensic genetics. Forensic practice sometimes requires the individualization of samples that may contain only highly degraded nuclear DNA, mitochondrial DNA or PCR inhibitors that hamper DNA amplification. We designed a new multiplex PCR with reduced size amplicons (<200 bp), providing a double sex determination (amelogenin plus two Y-STRs), the detection of two autosomal markers and the amplification of mitochondrial specific fragments from the hypervariable region I (HVI). Additionally, a quality sensor was developed to check for the presence of any PCR inhibitors. The new multiplex PCR shows a reproducible detection threshold down to 25 pg and gives signals even out of highly degraded materials. All signals are reproducible and reliable as it could be shown in comparison to results from commercially available STR multiplex-PCRs. In no case DNA fragments were detectable using any other assay when the quality sensor was not detectable. There was a good correlation between detection of mitochondrial specific fragments in the multiplex-PCR and success of subsequent sequencing of HVI region. The same could be shown for STR analysis: Most samples successfully analyzed in our PCR yielded at least a partial STR profile using a commercial STR kit. We present an assay that allows an easy, reliable, and cost efficient evaluation of DNA sample quality combined with a first rough sample individualization and sex determination suitable for forensic purposes. This assay may help the forensic lab personnel to decide on further sample processing. PMID:19215878

von Wurmb-Schwark, Nicole; Preusse-Prange, Andrea; Heinrich, Anke; Simeoni, Eva; Bosch, Thomas; Schwark, Thorsten

2009-01-07

92

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

PubMed

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

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

2013-07-18

93

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

PubMed Central

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

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

2011-01-01

94

Extraction and quantification of phosphorus derived from DNA and lipids in environmental samples.  

PubMed

Understanding the flux and turnover of phosphorus (P) in the environment is important due to the key role P plays in eutrophication and in the ambition to find cost-effective measures to mitigate it. Orthophosphate diesters, including DNA and phospholipids (PLs), represent a potentially degradable P pool that could support future primary production and eutrophication. In this study, extraction techniques were optimized and combined with colorimetric determination of extracted P to provide a selective quantification method for DNA-P and PL-P in agricultural soil, sediment and composted manure. The proposed method is rapid and reproducible with an RSD of <10%. Recovery, evaluated by spiking the sample matrices with DNA and PL standards, was over 95% for both DNA and PLs. The method can be used for the determination of the pool size of the two organic P fractions. Results show that DNA-P comprises 3.0% by weight of the total P (TP) content in the studied soil, 10.4% in the sediment and 8.4% in the compost samples. The values for PL-P are 0.5%, 6.0% and 1.7% for soil, sediment and compost, respectively. PMID:24054600

Paraskova, Julia V; Rydin, Emil; Sjöberg, Per J R

2013-05-24

95

Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil).  

PubMed

We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the number of DNA masses per petal at least doubled. This indicated chromatin fragmentation, either inside or outside the nucleus. Staining with the cationic lipophilic fluoroprobe DiOC6 indicated that each DNA mass had an external membrane. Fluorescence microscopy of the nuclei and DNA masses revealed an initial decrease in diameter together with chromatin condensation. The diameters of these condensed nuclei were about 70% of original. Two populations of nuclear diameter, one with an average diameter about half of the other, were observed at initial stages of nuclear fragmentation. The diameter of the DNA masses then gradually decreased further. The smallest observed DNA masses had a diameter less than 10% of that of the original nucleus. Cycloheximide treatment arrested the cytometrically determined changes in DNA fluorescence, indicating protein synthesis requirement. Ethylene inhibitors (AVG and 1-MCP) had no effect on the cytometrically determined DNA changes, suggesting that these processes are not controlled by endogenous ethylene. PMID:16738861

Yamada, Tetsuya; Takatsu, Yasumasa; Kasumi, Masakazu; Ichimura, Kazuo; van Doorn, Wouter G

2006-11-01

96

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

97

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

98

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

PubMed

Puparial cases are common remnants of necrophagous flies in crime investigations. They usually represent the longest developmental time and, therefore, they can be very useful for the estimation of the post-mortem interval (PMI). However, before any PMI estimate, it is crucial to identify the species of fly eclosed from each puparium associated with the corpse. Morphological characteristics of the puparium are often distinctive enough to permit a species identification. But, even an accurate morphological analysis of empty puparia cannot discriminate among different species of closely related flies. Furthermore, morphological identification may be impossible if the fly puparia are poorly preserved or in fragments. This study explores the applicability of biomolecular techniques on empty puparia and their fragments for identification purposes. A total of 63 empty puparia of necrophagous Diptera resulting from forensic casework were examined. Samples were divided into three groups according to size, type and time of eclosion in order to verify whether the physical characteristics and puparia weathering can influence the amount of DNA extraction. The results suggest that a reliable genetic identification of forensically important flies may also be performed from empty puparia and/or their fragments. However, DNA degradation can deeply compromise the genetic analysis since the older the fly puparia, the smaller are the amplified fragments. PMID:20031351

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

2010-01-19

99

Isolation of cholesterol- and deoxycholate-degrading bacteria from soil samples: evidence of a common pathway.  

PubMed

Nineteen different steroid-degrading bacteria were isolated from soil samples by using selective media containing either cholesterol or deoxycholate as sole carbon source. Strains that assimilated cholesterol (17 COL strains) were gram-positive, belonging to the genera Gordonia, Tsukamurella, and Rhodococcus, and grew on media containing other steroids but were unable to use deoxycholate as sole carbon source. Surprisingly, some of the COL strains unable to grow using deoxycholate as sole carbon source were able to catabolize other bile salts (e.g., cholate). Conversely, strains able to grow using deoxycholate as the sole carbon source (two DOC isolates) were gram-negative, belonging to the genus Pseudomonas, and were unable to catabolize cholesterol and other sterols. COL and DOC were included into the corresponding taxonomic groups based on their morphology (cells and colonies), metabolic properties (kind of substrates that support bacterial growth), and genetic sequences (16S rDNA and rpoB). Additionally, different DOC21 Tn5 insertion mutants have been obtained. These mutants have been classified into two different groups: (1) those affected in the catabolism of bile salts but that, as wild type, can grow in other steroids and (2) those unable to grow in media containing any of the steroids tested. The identification of the insertion point of Tn5 in one of the mutants belonging to the second group (DOC21 Mut1) revealed that the gene knocked-out encodes an A-ring meta-cleavage dioxygenase needed for steroid catabolism. PMID:22406861

Merino, E; Barrientos, A; Rodríguez, J; Naharro, G; Luengo, J M; Olivera, E R

2012-03-11

100

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

2012-11-28

101

DNA degradation in minicells of Escherichia coli K-12  

Microsoft Academic Search

The properties of minicell producing mutants of Escherichia coli deficient in gentic recombination were examined. Experiments were designed to test recombinant formation in conjugal crosses, survival following UV-irradiation in cells, and the state of DNA metabolism in minicells. The REC- phenotypes are unaffected by min+\\/- genotypes in whole cells. In contrast to minicells produced by rec+ parental cells, minicells from

George G. Khachatourians; M. C. Paterson; Ronald J. Sheehy; B. Van Dorp; T. E. Worthy

1975-01-01

102

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

PubMed

Although many genotoxic treatments upregulate the cyclin kinase inhibitor p21, agents such as UV irradiation trigger p21 degradation. This suggests that p21 blocks a process relevant for the cellular response to UV. Here, we show that forced p21 stabilization after UV strongly impairs damaged-DNA replication, which is associated with permanent deficiencies in the recruitment of DNA polymerases from the Y family involved in translesion DNA synthesis), with the accumulation of DNA damage markers and increased genomic instability. Remarkably, such noxious effects disappear when disrupting the proliferating cell nuclear antigen (PCNA) interacting motif of stable p21, thus suggesting that the release of PCNA from p21 interaction is sufficient to allow the recruitment to PCNA of partners (such as Y polymerases) relevant for the UV response. Expression of degradable p21 only transiently delays early replication events and Y polymerase recruitment after UV irradiation. These temporary defects disappear in a manner that correlates with p21 degradation with no detectable consequences on later replication events or genomic stability. Together, our findings suggest that the biological role of UV-triggered p21 degradation is to prevent replication defects by facilitating the tolerance of UV-induced DNA lesions. PMID:23723248

Mansilla, Sabrina F; Soria, Gastón; Vallerga, María Belén; Habif, Martín; Martínez-López, Wilner; Prives, Carol; Gottifredi, Vanesa

2013-05-30

103

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

PubMed Central

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

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

2013-01-01

104

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

PubMed Central

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

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

2002-01-01

105

Optimizing utilization of DNA from rare or archival anthropological samples.  

PubMed

There is widespread interest in obtaining genetic samples from human populations worldwide for various studies of human genetic diversity. Many samples exist today only in the form of small, rare, irreplaceable, or archival samples, such as material from ancient bone, hair bulbs, or remnants of samples collected in the field decades ago for the purpose of protein and blood type analysis. Here, we describe the application of an approach to amplify DNA, which we call adapter attachment and amplification (AAA). This approach is useful for amplifying the genome in a reasonably representative way from small amounts of starting material using standard PCR-based methods. We apply a version of these methods to DNA extracted from washed red blood cells collected in the 1960s and 1970s in the Amazon basin. AAA and similar approaches may make the analysis of archival samples possible without exhausting that irreplaceable material and may lead to greatly improved efficiency in collecting and using new anthropological genetic samples. PMID:8001910

Weiss, K M; Buchanan, A V; Daniel, C; Stoneking, M

1994-10-01

106

Direct multiplex sequencing (DMPS)--a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA  

PubMed Central

Although the emergence of high-throughput sequencing technologies has enabled whole-genome sequencing from extinct organisms, little progress has been made in accelerating targeted sequencing from highly degraded DNA. Here, we present a novel and highly sensitive method for targeted sequencing of ancient and degraded DNA, which couples multiplex PCR directly with sample barcoding and high-throughput sequencing. Using this approach, we obtained a 96% complete mitochondrial genome data set from 31 cave bear (Ursus spelaeus) samples using only two 454 Life Sciences (Roche) GS FLX runs. In contrast to previous studies relying only on short sequence fragments, the overlapping portion of our data comprises almost 10 kb of replicated mitochondrial genome sequence, allowing for the unambiguous differentiation of three major cave bear clades. Our method opens up the opportunity to simultaneously generate many kilobases of overlapping sequence data from large sets of difficult samples, such as museum specimens, medical collections, or forensic samples. Embedded in our approach, we present a new protocol for the construction of barcoded sequencing libraries, which is compatible with all current high-throughput technologies and can be performed entirely in plate setup.

Stiller, Mathias; Knapp, Michael; Stenzel, Udo; Hofreiter, Michael; Meyer, Matthias

2009-01-01

107

Direct multiplex sequencing (DMPS)--a novel method for targeted high-throughput sequencing of ancient and highly degraded DNA.  

PubMed

Although the emergence of high-throughput sequencing technologies has enabled whole-genome sequencing from extinct organisms, little progress has been made in accelerating targeted sequencing from highly degraded DNA. Here, we present a novel and highly sensitive method for targeted sequencing of ancient and degraded DNA, which couples multiplex PCR directly with sample barcoding and high-throughput sequencing. Using this approach, we obtained a 96% complete mitochondrial genome data set from 31 cave bear (Ursus spelaeus) samples using only two 454 Life Sciences (Roche) GS FLX runs. In contrast to previous studies relying only on short sequence fragments, the overlapping portion of our data comprises almost 10 kb of replicated mitochondrial genome sequence, allowing for the unambiguous differentiation of three major cave bear clades. Our method opens up the opportunity to simultaneously generate many kilobases of overlapping sequence data from large sets of difficult samples, such as museum specimens, medical collections, or forensic samples. Embedded in our approach, we present a new protocol for the construction of barcoded sequencing libraries, which is compatible with all current high-throughput technologies and can be performed entirely in plate setup. PMID:19635845

Stiller, Mathias; Knapp, Michael; Stenzel, Udo; Hofreiter, Michael; Meyer, Matthias

2009-07-27

108

DNA microarrays: sample quality control, array hybridization and scanning.  

PubMed

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 tested. 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 tissue. 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

2011-03-15

109

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

SciTech Connect

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

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

1984-01-01

110

Sensitive digital quantification of DNA methylation in clinical samples  

PubMed Central

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

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

2010-01-01

111

Involvement of phi29 DNA polymerase thumb subdomain in the proper coordination of synthesis and degradation during DNA replication.  

PubMed

Phi29 DNA polymerase achieves a functional coupling between its 3'-5' exonuclease and polymerization activities by means of important contacts with the DNA at both active sites. The placement and orientation of residues Lys538, Lys555, Lys557, Gln560, Thr571, Thr573 and Lys575 in a modelled phi29 DNA polymerase-DNA complex suggest a DNA-binding role. In addition, crystal structure of phi29 DNA polymerase-oligo (dT)5 complex showed Leu567, placed at the tip of the thumb subdomain, lying between the two 3'-terminal bases at the exonuclease site. Single replacement of these phi29 DNA polymerase residues by alanine was made, and mutant derivatives were overproduced and purified to homogeneity. The results obtained in the assay of their synthetic and degradative activities, as well as their coordination, allow us to propose: (1) a primer-terminus stabilization role at the polymerase active site for residues Lys538, Thr573 and Lys575, (2) a primer-terminus stabilization role at the exonuclease active site for residues Leu567 and Lys555 and (3) a primer-terminus binding role in both editing and polymerization modes for residue Gln560. The results presented here lead us to propose phi29 DNA polymerase thumb as the main subdomain responsible for the coordination of polymerization and exonuclease activities. PMID:16757576

Pérez-Arnaiz, Patricia; Lázaro, José M; Salas, Margarita; de Vega, Miguel

2006-06-06

112

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

PubMed

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

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

2013-05-23

113

Bacterial argonaute samples the transcriptome to identify foreign DNA.  

PubMed

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

2013-09-12

114

Pulsed-Field Gel Electrophoresis Study of Mycobacterium abscessus Isolates Previously Affected by DNA Degradation  

Microsoft Academic Search

DNA degradation (which results in a smear pattern) occurs with almost 50% of Mycobacterium abscessus strains during pulsed-field gel electrophoresis (PFGE). We assessed the potential benefit of using thiourea- containing buffer with M. abscessus by studying 69 isolates not previously typeable by PFGE (i.e., those with a smear pattern). Random (epidemiologically unrelated) isolates that were typeable (no smear pattern) were

Yansheng Zhang; Mitchell A. Yakrus; Edward A. Graviss; Natalie Williams-Bouyer

115

Mapping of an adenovirus function involved in the inhibition of DNA degradation.  

PubMed Central

A function involved in the inhibition of DNA degradation has been assigned through complementation tests to a product of region E1b of the adenovirus genome (between 4.5 and 10.5 map units). DNA degradation induced by the adenovirus type 12 (Ad12) cyt mutant H12cyt70 and the Ad5 early deletion mutant dl313 (with the deletion between 3.5 and 10.7 map units) was inhibited by coinfection with Ad5 region E1a (between 0 and 4.5 map units) mutants dl312 and hr1 and region E1b mutant hr6. The defect of inhibition of DNA degradation in Ad5 dl313 was also complemented in 293 cells. This DNase-inhibitory function does not appear to involve polypeptide IX or the 58,000-dalton polypeptide. Wild-type Ad12 induced DNA degradation in hamster embryo cells, suggesting that the DNase-inhibitory function is not expressed in these nonpermissive cells. Additional evidence suggests the involvement of a second viral product which positively influences the DNase activity and which appears to be an early function. Images

Lai Fatt, R B; Mak, S

1982-01-01

116

The Development of Reduced Size STR Amplicons as Tools for Analysis of Degraded DNA  

Microsoft Academic Search

New multiplex PCR sets of commonly used short tandem repeat (STR) markers have been developed to produce PCR products that are reduced in size when compared to standard commercial STR kits. The reduction in size of these amplicons can facilitate the examination and analysis of degraded DNA evidence by improving amplification efficiency. This \\

John M. Butler; Yin Shen; Bruce R. McCord

117

The Roles and Acting Mechanism of Caenorhabditis elegans DNase II Genes in Apoptotic DNA Degradation and Development  

Microsoft Academic Search

DNase II enzymes are acidic endonucleases that have been implicated in mediating apoptotic DNA degradation, a critical cell death execution event. C. elegans genome contains three DNase II homologues, NUC-1, CRN-6, and CRN-7, but their expression patterns, acting sites, and roles in apoptotic DNA degradation and development are unclear. We have conducted a comprehensive analysis of three C. elegans DNase

Huey-Jen Lai; Szecheng J. Lo; Eriko Kage-Nakadai; Shohei Mitani; Ding Xue

2009-01-01

118

Saliva sampling in global clinical studies: the impact of low sampling volume on performance of DNA in downstream genotyping experiments.  

PubMed

BACKGROUND: The collection of viable DNA samples is an essential element of any genetics research programme. Biological samples for DNA purification are now routinely collected in many studies with a variety of sampling methods available. Initial observation in this study suggested a reduced genotyping success rate of some saliva derived DNA samples when compared to blood derived DNA samples prompting further investigation. METHODS: Genotyping success rate was investigated to assess the suitability of using saliva samples in future safety and efficacy pharmacogenetics experiments. The Oragene(R) OG-300 DNA Self-Collection kit was used to collect and extract DNA from saliva from 1468 subjects enrolled in global clinical studies. Statistical analysis evaluated the impact of saliva sample volume of collection on the quality, yield, concentration and performance of saliva DNA in genotyping assays. RESULTS: Across 13 global clinical studies that utilized the Oragene(R) OG-300 DNA Self-Collection kit there was variability in the volume of saliva sample collection with ~31% of participants providing 0.5 mL of saliva, rather than the recommended 2 mL. While the majority of saliva DNA samples provided high quality genotype data, collection of 0.5 mL volumes of saliva contributed to DNA samples being significantly less likely to pass genotyping quality control standards. Assessment of DNA sample characteristics that may influence genotyping outcomes indicated that saliva sample volume, DNA purity and turbidity were independently associated with sample genotype pass rate, but that saliva collection volume had the greatest effect. CONCLUSION: When employing saliva sampling to obtain DNA, it is important to encourage all study participants to provide sufficient sample to minimize potential loss of data in downstream genotyping experiments. PMID:23759220

Pulford, David J; Mosteller, Michael; Briley, J David; Johansson, Kelley W; Nelsen, Anita J

2013-06-10

119

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

PubMed

DNA is known to be aggregated by metal ions including Mn(2+) ions, but analysis of the aggregation process from a chemical viewpoint, which means identification of the product yielded during the process, has not been performed yet. On examination of the kinds of degraded materials that were in the supernatant obtained on centrifugation of a DNA mixture aggregated under conditions of 10 mM Mn(2+) ions ([Mn]/[P] = 46.3) at 70 degrees C for 1 h, the degradation products were found to be dAMP, dCMP, dGMP, and TMP. These dNMPs were purified by HPLC on TSKgel ODS-80Ts and identified by LC-TOF/MS. The degradation activity was lost on pretreatment of the DNA with a phenol-chloroform mixture, and the activity was recovered by pretreatment with a mixture of DMSO and a buffer containing surfactants. Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), and Cd(2+), as transition element metal ions, were effective as to the degradation into dNMP. Mg(2+), Ca(2+), Sr(2+), and Ba(2+), as alkali earth element metal ions, were not effective as to the degradation. Monovalent anions such as Cl(-), CH(3)OO(-), and NO(3)(-) were found to increase the degradation rate. Sixty mug of the 120 mug of the starting DNA in 450 mul was degraded into dNMP on reaction for 1 h in the presence of 100 mM NaCl and 10 mM Mn(2+) ions. In this process, aggregation did not occur, and thus was not considered to be necessary for degradation. The degradation was found not to occur at pH 7.0, and to be very sensitive to pH. The OH(-) ion should have a critical role in cleavage of the phosphodiester linkages in this case. The dNMP obtained in the degradation process was found to be only 5'-NMP, based on the H(1)NMR spectra. This prosess should prove to be a new process for the production of 5'-dNMP in addtion to the exonuclease. PMID:17986770

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

2007-11-07

120

ZEN1 Is a Key Enzyme in the Degradation of Nuclear DNA during Programmed Cell Death of Tracheary Elements  

Microsoft Academic Search

Tracheary elements (TEs) have a unique cell death program in which the rapid collapse of the vacuole triggers the be- ginning of nuclear degradation. Although various nucleases are known to function in nuclear DNA degradation in ani- mal apoptosis, it is unclear what hydrolase is involved in nuclear degradation in plants. In this study, we demonstrated that an S1-type nuclease,

Jun Ito; Hiroo Fukuda

2002-01-01

121

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

PubMed Central

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

2012-01-01

122

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

123

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.

Zhang, Lifen; Chen, Zhenzhen; Li, Yanfeng

2013-01-01

124

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

PubMed

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

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

2005-12-28

125

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

2012-03-07

126

Nuclear cataract caused by a lack of DNA degradation in the mouse eye lens.  

PubMed

The eye lens is composed of fibre cells, which develop from the epithelial cells on the anterior surface of the lens. Differentiation into a lens fibre cell is accompanied by changes in cell shape, the expression of crystallins and the degradation of cellular organelles. The loss of organelles is believed to ensure the transparency of the lens, but the molecular mechanism behind this process is not known. Here we show that DLAD ('DNase II-like acid DNase', also called DNase IIbeta) is expressed in human and murine lens cells, and that mice deficient in the DLAD gene are incapable of degrading DNA during lens cell differentiation--the undigested DNA accumulates in the fibre cells. The DLAD-/- mice develop cataracts of the nucleus lentis, and their response to light on electroretinograms is severely reduced. These results indicate that DLAD is responsible for the degradation of nuclear DNA during lens cell differentiation, and that if DNA is left undigested in the lens, it causes cataracts of the nucleus lentis, blocking the light path. PMID:12944971

Nishimoto, Sogo; Kawane, Kohki; Watanabe-Fukunaga, Rie; Fukuyama, Hidehiro; Ohsawa, Yoshiyuki; Uchiyama, Yasuo; Hashida, Noriyasu; Ohguro, Nobuyuki; Tano, Yasuo; Morimoto, Takeshi; Fukuda, Yutaka; Nagata, Shigekazu

2003-08-28

127

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

PubMed Central

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

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

2011-01-01

128

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

PubMed

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

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

2013-06-20

129

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

PubMed

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

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

2013-08-01

130

Atomic Force Microscopy Of Uncoated Plasmid DNA: Nanometer Resolution with only Nanogram Amounts of Sample  

Microsoft Academic Search

Reproducible, high-contrast, nanometer-resolution AFM images of uncoated plasmid DNA can be obtained with nanogram quantities of DNA with the help of two advances in sample preparation: (1) Heating a DNA solution at 35 °C for 10 to 20 minutes before deposition on mica helps separate and spread the DNA and (2) Using 5 microliter drops of the heated DNA solution

Min-Qian Li; Helen G. Hansma; James Vesenka; Gregory Kelderman; Paul K. Hansma

1992-01-01

131

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

PubMed Central

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

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

2010-01-01

132

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

PubMed

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

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

2010-09-15

133

DNA degradation in the kidney of folic acid-treated guinea pigs.  

PubMed

Previous investigators agree on the increased DNA synthesis and destruction of tissues caused by folic acid (FA) administered parenterally. This study aims to clarify whether DNA degradation due to the destruction of cells and nuclei precedes DNA synthesis following FA administration. Forty guinea pigs were divided into four groups: group 1, contained 10 controls; in group 2, ten animals received intraperitoneally 300 mg/kg of body wt FA; in group 3, ten animals received FA and 12 h later frusemide intramuscularly in a dose of 7 mg/kg body wt; and finally in group 4, ten animals received frusemide as in group 3. FA produced necrosis of the epithelial cells of the convoluted tubules as the detection of the beta-aminoisobutyric acid end product of DNA and thymine catabolism indicated. Frusemide administered in group 3 had a favourable effect on the acute renal failure induced by FA. PMID:10885507

Zeis, P M; Tzaki, M; Nakopoulou, L; Nicolaidou, P; Kavazarakis, E; Messaritaki, A; Moustaki, M; Zeis, M P; Gourgiotis, D

2000-01-01

134

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

PubMed Central

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

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

2012-01-01

135

Intracellular delivery of DNA and enzyme in active form using degradable carbohydrate-based nanogels.  

PubMed

The facile encapsulation of biomolecules along with efficient formulation and storage makes nanogels ideal candidates for drug and gene delivery. So far, nanogels have not been used for the codelivery of plasmid DNA and proteins due to several limitations, including low encapsulation efficacy of biomolecule of similar charges and the size of cargo materials. In this study, temperature and pH sensitive carbohydrate-based nanogels are synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization technique and are studied in detail for their capacity to encapsulate and codeliver plasmid DNA and proteins. The temperature sensitive property of nanogels allows the facile encapsulation of biomaterials, while its acid-degradable profile allows the burst release of biomolecules in endosomes. Hence these materials are expected to serve as efficient vectors to deliver biomolecules of choice either alone or as codelivery system. The nanogels produced are relatively monodisperse and are around 30-40 nm in diameter at 37 °C. DNA condensation efficacy of the nanogels is dependent on the hydrophobic property of the core of the nanogels. The DNA-nanogel complexes are formed by the interaction of carbohydrate residues of nanogels with the DNA, and complexes are further stabilized with linear cationic glycopolymers. The DNA-nanogels complexes are also studied for their protein loading capacity. The degradation of the nanogels and the controlled release of DNA and proteins are then studied in vitro. Furthermore, the addition of a nontoxic, cationic glycopolymer to the nanogel-DNA complexes is found to improve the cellular uptake and hence to improve gene expression. PMID:22970989

Ahmed, Marya; Narain, Ravin

2012-09-25

136

A rapid wire-based sampling method for DNA profiling.  

PubMed

This paper reports the results of a commission to develop a field deployable rapid short tandem repeat (STR)-based DNA profiling system to enable discrimination between tissues derived from a small number of individuals. Speed was achieved by truncation of sample preparation and field deployability by use of an Agilent 2100 Bioanalyser(TM). Human blood and tissues were stabbed with heated stainless steel wire and the resulting sample dehydrated with isopropanol prior to direct addition to a PCR. Choice of a polymerase tolerant of tissue residues and cycles of amplification appropriate for the amount of template expected yielded useful profiles with a custom-designed quintuplex primer set suitable for use with the Bioanalyser(TM). Samples stored on wires remained amplifiable for months, allowing their transportation unrefrigerated from remote locations to a laboratory for analysis using AmpFlSTR(®) Profiler Plus(®) without further processing. The field system meets the requirements for discrimination of samples from small sets and retains access to full STR profiling when required. PMID:22211864

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

2011-12-28

137

Laser irradiation and Raman spectroscopy of single living cells and chromosomes: Sample degradation occurs with 514. 5 nm but not with 660 nm laser light  

SciTech Connect

In Raman spectroscopic measurements of single cells (human lymphocytes) and chromosomes, using a newly developed confocal Raman microspectrometer and a laser excitation wavelength of 514.5 nm, degradation of the biological objects was observed. In the experiments high power microscope objectives were used, focusing the laser beam into a spot {approximately} 0.5 micron in diameter. At the position of the laser focus a paling of the samples became visible even when the laser power on the sample was reduced to less than 1 mW. This was accompanied by a gradual decrease in the intensity of the Raman signal. With 5 mW of laser power the events became noticeable after a period of time in the order of minutes. It is shown that a number of potential mechanisms, such as excessive sample heating due to absorption of laser light, multiple photon absorption, and substrate heating are unlikely to play a role. In experiments with DNA solutions and histone protein solutions no evidence of photo damage was found using laser powers up to 25 mW. No degradation of cells and chromosomes occurs when laser light of 660 nm is used. The most plausible explanation therefore seems to be that the sample degradation is the result of photochemical reactions initiated by laser excitation at 514.5 nm of as yet unidentified sensitizer molecules or complexes present in chromosomes and cells but not in purified DNA and histone protein samples.

Puppels, G.J.; Olminkhof, J.H.; Segers-Nolten, G.M.; Otto, C.; de Mul, F.F.; Greve, J. (Department of Applied Physics, University of Twente, Enschede (Netherlands))

1991-08-01

138

Enzymatic degradation of starch thermoplastic blends using samples of different thickness.  

PubMed

The material studied was a thermoplastic blend of corn starch with a poly(ethylene-vinyl alcohol) copolymer, SEVA-C. The influence of both the material's exposed surface and enzyme concentration on degradation kinetics was studied. As alpha-amylase is present in the blood plasma, experiments were performed, varying the material thickness and the alpha-amylase between 50 and 100 units/l, at 37 degrees C, lasting up to 90 days. Four different batches using SEVA-C and starch samples of different thickness were performed. The positive correlation between degradation rate and the exposed material surface was confirmed, since thin films with larger exposed surfaces were degraded faster than thick square plates having the same total mass. The degradation extent depends on the total amount of amorphous starch present in the formulation rather than on the amount of enzyme used and the minimum thickness to ensure maximum degradation was estimated to be close to 0.25 mm. PMID:18853238

Araújo, M Alberta; Cunha, António M; Mota, Manuel

2008-10-14

139

Degradation of BRCA2 in Alkyltransferase-Mediated DNA Repair and Its Clinical Implications  

Microsoft Academic Search

Germ-line mutations in BRCA2 have been linked to early-onset familial breast cancer. BRCA2 is known to play a key role in repairing double-strand breaks. Here,we describe the involvement of BRCA2 in O6-alkylguanine DNA alkyltransferase (AGT)-mediated repair of O 6-methylguanine adducts. We show that BRCA2 physically associates and undergoes repair- mediated degradation with AGT. In contrast,BRCA2 with a 29-amino-acid deletion in

Subha Philip; Srividya Swaminathan; Sergey G. Kuznetsov; Sreenivas Kanugula; Kajal Biswas; Suhwan Chang; Natalia A. Loktionova; Diana C. Haines; Philipp Kaldis; Anthony E. Pegg; Shyam K. Sharan

2008-01-01

140

Soil colloids-bound plasmid DNA: Effect on transformation of E. coli and resistance to DNase I degradation  

Microsoft Academic Search

The adsorption and binding of plasmid p34S DNA on four different colloidal fractions from a Brown soil and clay minerals in the presence of various Ca2+ concentrations, the ability of bound DNA to transform competent cells of CaCl2-treated Escherichia coli, and the resistance of bound DNA to degradation by DNase I were studied. DNA adsorption on soil colloids and clay

P. Cai; Q. Huang; W. Chen; D. Zhang; K. Wang; D. Jiang; W. Liang

2007-01-01

141

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

PubMed Central

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

Walia, S; Khan, A; Rosenthal, N

1990-01-01

142

SCFbetaTrCP-mediated degradation of Claspin regulates recovery from the DNA replication checkpoint response.  

PubMed

During replicative stress, Claspin mediates the phosphorylation and consequent activation of Chk1 by ATR. We found that during recovery from the DNA replication checkpoint response, Claspin is degraded in a betaTrCP-dependent manner. In vivo, Claspin is phosphorylated in a canonical DSGxxS degron sequence, which is typical of betaTrCP substrates. Phosphorylation of Claspin is mediated by Plk1 and is essential for binding to betaTrCP. In vitro ubiquitylation of Claspin requires betaTrCP, Plk1, and an intact DSGxxS degron. Significantly, expression of a stable Claspin mutant unable to bind betaTrCP prolongs the activation of Chk1, thereby attenuating the recovery from the DNA replication stress response and significantly delaying entry into mitosis. Thus, the SCFbetaTrCP-dependent degradation of Claspin is necessary for the efficient and timely termination of the DNA replication checkpoint. Importantly, in response to DNA damage in G2, Claspin proteolysis is inhibited to allow the prompt reestablishment of the checkpoint. PMID:16885022

Peschiaroli, Angelo; Dorrello, N Valerio; Guardavaccaro, Daniele; Venere, Monica; Halazonetis, Thanos; Sherman, Nicholas E; Pagano, Michele

2006-08-01

143

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.

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

2013-01-01

144

Green Fluorescent Protein-Based Biosensor To Detect and Quantify Stress Responses Induced by DNA-Degrading Colicins ? †  

PubMed Central

Here we report the development of a whole-cell biosensor to detect and quantify the induction of the SOS response activated by DNA-degrading colicins. This biosensor utilizes the SOS-responsive cda promoter to regulate the expression of green fluorescent protein. The biosensor assay revealed induction of stress for all DNA-degrading reference colicins (E2, E7, and E8).

Abraham, Sam; Chin, James; Brouwers, Huub J. M.; Turner, Bernadette; Zhang, Ren; Chapman, Toni A.

2011-01-01

145

Comparison of the lignin-degrading white rot fungi Phanerochaete chrysosporium and Sporotrichum pulverulentum at the DNA level  

Microsoft Academic Search

DNA-hybridisation studies showed a close relationship between Phanerochaete chrysosporium ME446, most used in lignin degradation studies, and Sporotrichum pulverulentum Novobranova, the other standard lignin degrading strain. Two other strains of P. chrysosporium were both less related. We show that P. chrysosporium ME446 and S. pulverulentum Novobranova both have a GC-content of 59% for chromosomal DNA with the rRNA genes present

Ute Raeder; Paul Broda

1984-01-01

146

Degradation of p12 Subunit by CRL4Cdt2 E3 Ligase Inhibits Fork Progression after DNA Damage.  

PubMed

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

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

2013-09-10

147

Estrogenic activity of bio-degradation products of C-heavy oil revealed by gene-expression profiling using an oligo-DNA microarray system.  

PubMed

Degradation of heavy oil by bacteria to decompose organic compounds such as aliphatic and aromatic hydrocarbons has been used in bioremediation. However, the biological and environmental effects of the degradation products including intermediates are still not clear. Here, we monitored the degradation of C-heavy oil by analyzing the products formed in cultures with oil-degrading bacteria (complex microbes or a single bacterial strain). Furthermore, proliferation assays using breast cancer MCF-7 cells and gene-expression profiling of MCF-7 cells using oligonucleotide-DNA microarrays were performed to evaluate the estrogenic activity of the degradation products. While the products did not show any significant cell-proliferative activity, the oil samples cultured for longer periods (2-3 months), whether cultured with mixed microbes or a single bacterial strain, showed gene-expression profiles similar to that of 17?-estradiol (E2). These results suggest that oil-degradation products have estrogenic activity, and estrogen-like components could possibly be produced during the degradation process. PMID:22580234

Zhu, Yun; Kitamura, Keiko; Maruyama, Akihiko; Higashihara, Takanori; Kiyama, Ryoiti

2012-05-11

148

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

PubMed

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

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

2013-05-19

149

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

PubMed

Segmental progeroid syndromes are groups of disorders with multiple features suggestive of accelerated aging. One subset of adult-onset progeroid syndromes, referred to as atypical Werner syndrome, is caused by mutations in the LMNA gene, which encodes a class of nuclear intermediate filaments, lamin A/C. We previously described rapid telomere attrition and accelerated replicative senescence in cultured fibroblasts overexpressing mutant lamin A. In this study, we investigated the cellular phenotypes associated with accelerated telomere shortening in LMNA mutant primary fibroblasts. In early passage primary fibroblasts with R133L or L140R LMNA mutations, shelterin protein components were already reduced while cells still retained telomere lengths comparable to those of controls. There was a significant inverse correlation between the degree of abnormal nuclear morphology and the level of TRF2, a shelterin subunit, suggesting a potential causal relationship. Stabilization of the telomeres via the introduction of the catalytic subunit of human telomerase, hTERT (human telomerase reverse transcriptase), did not prevent degradation of shelterin components, indicating that reduced TRF2 in LMNA mutants is not mediated by short telomeres. Interestingly, ?-H2AX foci (reflecting double strand DNA damage) in early passage LMNA mutant primary fibroblasts and LMNA mutant hTERT fibroblasts were markedly increased in non-telomeric regions of DNA. Our results raise the possibility that mutant lamin A/C causes global genomic instability with accumulation of non-telomeric DNA damage as an early event, followed by TRF2 degradation and telomere shortening. PMID:23847654

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

2013-07-05

150

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

PubMed Central

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

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

2013-01-01

151

Degradation and possible carry over of feed DNA monitored in pigs and poultry  

Microsoft Academic Search

A possible carry over of foreign food DNA into the body after consumption was examined. After feeding pigs with conventional and recombinant (Bt-) maize, different body samples were investigated using DNA-extraction followed by PCR procedures to detect chloroplast genes of different length (199 bp and 532 bp), a maize-specific gene (zein) and a specific transgene present in Bt-maize (cryIa). Initially,

Andreas Klotz; Johann Mayer; Ralf Einspanier

2002-01-01

152

DNA-SIP reveals that Syntrophaceae play an important role in methanogenic hexadecane degradation.  

PubMed

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

2013-07-01

153

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

PubMed Central

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

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

2013-01-01

154

The anthocyanidin delphinidin mobilizes endogenous copper ions from human lymphocytes leading to oxidative degradation of cellular DNA  

Microsoft Academic Search

Epidemiological and experimental evidence exists to suggest that pomegranate and its juice possess chemopreventive and anticancer properties. The anthocyanidin delphinidin is a major polyphenol present in pomegranates and has been shown to be responsible for these effects. Plant polyphenols are recognized as naturally occurring antioxidants but also catalyze oxidative DNA degradation of cellular DNA either alone or in the presence

Sarmad Hanif; Uzma Shamim; M. F. Ullah; Asfar S. Azmi; Showket H. Bhat; S. M. Hadi

2008-01-01

155

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

PubMed

The PrepFiler Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 microL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA paper), and touch evidence-type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol-chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts. PMID:19302383

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

2009-03-16

156

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

PubMed

Extracellular or cell free DNA has been found to exist in many biological media such as blood and saliva. To check whether cell free DNA is present in the supernatant which is normally discarded during several DNA extraction processes, such as Chelex(®) extraction, DNA profiles of cell pellet and concentrated supernatant from 30 artificial case like samples and from 100 real forensic samples were compared. Presence of cell free DNA was shown in all investigated sample types. Moreover, in some samples additional alleles, not detected during analysis of the cell pellet, were detected, offering valuable information which would normally have been discarded together with the supernatant. The results presented here indicate that cell free DNA deserves further consideration since it has the potential to increase the DNA yield in forensic casework samples in general and in contact traces in particular. PMID:23318134

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

2013-01-11

157

An Isothermal Method for Whole Genome Amplification of Fresh and Degraded DNA for Comparative Genomic Hybridization, Genotyping and Mutation Detection  

Microsoft Academic Search

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and

Cheryl I. P. Lee; Siew Hong Leong; Adrian E. H. Png; Keng Wah Choo; Christopher Syn; Dennis T. H. Lim; Hai Yang Law; Oi Lian Kon

2006-01-01

158

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

PubMed Central

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

2012-01-01

159

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

160

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

PubMed

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

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

2012-11-29

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

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

PubMed

The characterization of externally visible traits by DNA analysis is already an important tool when investigating ancient skeletal remains and may gain similar importance in future forensic DNA analysis. This, however, depends on the different legal regulations in the different countries. Besides eye or hair color, the population origin can provide crucial information in criminal prosecution. In this study, we present the analysis of 16 single-nucleotide polymorphisms (SNPs) combined to two robust SNaPshot assays with a detection threshold of 25-pg DNA. This assay was applied to 891 people from seven different populations (West Africa, North Africa, Turkey, Near East, Balkan states, North Europe, and Japan) with a thorough statistical evaluation. The prediction model was validated by an additional 125 individuals predominantly with an ancestry from those same regions. The specificity of these SNPs for the prediction of all population origins is very high (>90 %), but the sensitivity varied greatly (more than 90 % for West Africa, but only 25 % for the Near East). We could identify West Africans with a certainty of 100 %, and people from North Africa, the Balkan states, or North Europe nearly with the same reliability while determination of Turks or people from the Near East was rather difficult. In conclusion, the two SNaPshot assays are a powerful and reliable tool for the identification of people with an ancestry in one of the above listed populations, even from degraded DNA. PMID:22918435

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

2012-08-24

163

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.

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

2011-01-01

164

DNA sexing in Humboldt Penguins (Spheniscus humboldti) from feather samples.  

PubMed

Humboldt Penguins (Spheniscus humboldti) show little sexual dimorphism, and although males are usually heavier and larger than females, sexing by direct observation may be difficult, especially in young subjects. In this paper we evaluate the utility of the molecular approach, for sexing impuberal Humboldt Penguins from feathers. Firstly, a PCR test was used employing primers that amplify the homologous region of the CHD-W gene, unique in female, and the CHD-Z gene, occurring in the two sexes. The analysis of the PCR products showed a band of 370 bp in males and two bands of 370 and 380 bp in females. Additionally, to confirm these results, the PCR products were digested with HaeIII and Asp700 for RFLP analysis. Male PCR products showed two bands (310 and 60 bp) after digestion with HaeIII, and a unique band (370 bp) using Asp700, while all fragments obtained from females resolved into three bands using both HaeIII (380, 310 and 60 bp) and Asp700 (370, 270 and 110 bp), confirming the previous PCR sex determination. Results from these two different DNA-based tests were in accordance, in all cases, with sexes checked by preliminary cloacoscopy. Thus, it was found that the PCR method from feather samples alone is sufficient, reliable and without any risks for a rapid sexing in Humboldt Penguin. This non-invasive sexing technique can be useful at any age to verify the sex ratio in field populations and for gender identification in ex situ conservation programs. PMID:18258392

Costantini, V; Guaricci, A C; Laricchiuta, P; Rausa, F; Lacalandra, G M

2008-01-03

165

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

NASA Astrophysics Data System (ADS)

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

Saenger, K. L.

2012-10-01

166

Comparative analysis of microbial DNA extraction protocols for groundwater samples.  

PubMed

A comparative analysis of four different DNA extraction protocols was performed to determine the best choice for groundwater microbial diversity studies using temperature gradient gel electrophoresis (TGGE) analysis. The methods used were a chelex-based method, a modified salting out procedure (MSOP), and the commercial kits Epicentre and FastDNA. Both commercial kits exhibited the greatest reproducibility in their methods; however, their band patterns were very different. The protocol that showed the highest diversity was the chelex-based method, and the one that showed the lowest diversity was the FastDNA kit. PMID:21683680

Purswani, Jessica; Martín-Platero, Antonio Manuel; Reboleiro-Rivas, Patricia; González-López, Jesús; Pozo, Clementina

2011-05-27

167

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

168

Highly Effective DNA Extraction Method from Fresh, Frozen, Dried and Clotted Blood Samples  

PubMed Central

Introduction Today, with the tremendous potential of genomics and other recent advances in science, the role of science to improve reliable DNA extraction methods is more relevant than ever before. The ideal process for genomic DNA extraction demands high quantities of pure, integral and intact genomic DNA (gDNA) from the sample with minimal co-extraction of inhibitors of downstream processes. Here, we report the development of a very rapid, less-hazardous, and high throughput protocol for extracting of high quality DNA from blood samples. Methods Dried, clotted and ethylene diamine tetra-acetic acid (EDTA) treated fresh and frozen blood samples were extracted using this method in which the quality and integrity of the extracted DNA were corroborated by agarose gel electrophoresis, PCR reaction and DNA digestion using restricted enzyme. The UV spectrophotometric and gel electrophoresis analysis resulted in high A260/A280 ratio (>1.8) with high intactness of DNA. Results PCR and DNA digestion experiments indicated that the final solutions of extracted DNA contained no inhibitory substances, which confirms that the isolated DNA is of good quality. Conclusion The high quality and quantity of current method, no enzymatic processing and accordingly its low cost, make it appropriate for DNA extraction not only from human but also from animal blood samples in any molecular biology labs.

Samadi Shams, Sara; Zununi Vahed, Sepideh; Soltanzad, Farzaneh; Kafil, Vala; Barzegari, Abolfazl; Atashpaz, Sina; Barar, Jaleh

2011-01-01

169

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

Microsoft Academic Search

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 \\

Julie T. Millard; André M. Pilon

2003-01-01

170

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

PubMed Central

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

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

2013-01-01

171

The anthocyanidin delphinidin mobilizes endogenous copper ions from human lymphocytes leading to oxidative degradation of cellular DNA.  

PubMed

Epidemiological and experimental evidence exists to suggest that pomegranate and its juice possess chemopreventive and anticancer properties. The anthocyanidin delphinidin is a major polyphenol present in pomegranates and has been shown to be responsible for these effects. Plant polyphenols are recognized as naturally occurring antioxidants but also catalyze oxidative DNA degradation of cellular DNA either alone or in the presence of transition metal ions such as copper. In this paper we show that similar to various other classes of polyphenols, delphinidin is also capable of causing oxidative degradation of cellular DNA. Lymphocytes were exposed to various concentrations of delphinidin (10, 20, 50 microM) for 1h and the DNA breakage was assessed using single cell alkaline gel electrophoresis (Comet assay). Inhibition of DNA breakage by several scavengers of reactive oxygen species (ROS) indicated that it is caused by the formation of ROS. Incubation of lymphocytes with neocuproine (a cell membrane permeable Cu(I) chelator) inhibited DNA degradation in intact lymphocytes in a dose dependent manner. Bathocuproine, which is unable to permeate through the cell membrane, did not cause such inhibition. We have further shown that delphinidin is able to degrade DNA in cell nuclei and that such DNA degradation is also inhibited by neocuproine suggesting that nuclear copper is mobilized in this reaction. These results indicate that the generation of ROS possibly occurs through mobilization of endogenous copper ions. The results are in support of our hypothesis that the prooxidant activity of plant polyphenols may be an important mechanism for their anticancer properties. PMID:18486296

Hanif, Sarmad; Shamim, Uzma; Ullah, M F; Azmi, Asfar S; Bhat, Showket H; Hadi, S M

2008-04-09

172

Influence of sampling practices on the appearance of DNA image histograms of prostate cells in FNAB samples.  

PubMed

Twenty-one fine needle aspiration biopsies (FNAB) of the prostate, diagnostically classified as definitely malignant, were studied. The Papanicolaou or H&E stained samples were destained and then stained for DNA with the Feulgen reaction. DNA cytometry was applied after different sampling rules. The histograms varied according to the sampling rule applied. Because free cells between cell groups were easier to measure than cells in the cell groups, two sampling rules were tested in all samples: (i) cells in the cell groups were measured, and (ii) free cells between cell groups were measured. Abnormal histograms were more common after the sampling rule based on free cells, suggesting that abnormal patterns are best revealed through the free cells in these samples. The conclusions were independent of the applied histogram interpretation method. PMID:10468406

Buhmeida, A; Kuopio, T; Collan, Y

1999-01-01

173

UVC-induced mitochondrial degradation via autophagy correlates with mtDNA damage removal in primary human fibroblasts.  

PubMed

Mitochondrial DNA (mtDNA) is more susceptible than nuclear DNA to helix-distorting damage via exposure to environmental genotoxins, partially due to a lack of nucleotide excision repair. Thus, this damage is irreparable and persistent in mtDNA in the short term. We recently found that helix-distorting mtDNA damage induced by ultraviolet C radiation (UVC) is gradually removed in Caenorhabditis elegans and that removal is dependent upon autophagy and mitochondrial dynamics. We here report the effects of UVC exposure on mitophagy, mitochondrial morphology, and indicators of mitochondrial function in mammalian cells. Exposure to UVC induced autophagy within 24 h; nonetheless, significant mitochondrial degradation was not observed until 72 h post exposure. Mitochondrial mass, morphology, and function were not significantly altered. These data further support the idea that persistent mtDNA damage is removed by autophagy and also suggest a powerful compensatory capacity for dealing with mtDNA damage. PMID:23132756

Bess, Amanda S; Ryde, Ian T; Hinton, David E; Meyer, Joel N

2012-11-06

174

Bleomycin mediated degradation of DNA-RNA hybrids does not involve C-1' chemistry.  

PubMed Central

Incubation of Fe(II) bleomycin and O2 with a number of 'A'-like DNA-RNA hybrid homopolymers at 4 atm O2 results in formation of base propenal and base in a ratio of approximately 1.0:1.0. This ratio differs dramatically from the corresponding ratio of approximately 10:1.0 observed when activated BLM degrades 'B'-like DNA homopolymers. Experiments were undertaken to determine if the shift to enhanced base production observed in the A-like hybrids is the result of C-1' chemistry in addition to the C-4' chemistry normally observed with B-like DNA under identical conditions. Increased accessibility of the 1'-hydrogen might be anticipated due to widening of the minor groove in the A-like conformers. Experiments using poly([1'-3H]dA) poly(rU) and poly([U-14C]dA) poly(rU) indicated that neither 3H2O nor deoxyribonolactone accompanied adenine release. In addition, studies using poly([4'-2H]dA) poly(rU) and poly([1'-2H]dA) poly(rU) unambiguously establish that the altered base to base propenal ratio is not the result of C-1' chemistry, but a direct consequence of C-4' chemistry.

Absalon, M J; Krishnamoorthy, C R; McGall, G; Kozarich, J W; Stubbe, J

1992-01-01

175

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

2011-04-26

176

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

PubMed

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

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

2010-06-01

177

Cloning of a cDNA encoding an ectoenzyme that degrades thyrotropin-releasing hormone.  

PubMed Central

Thyrotropin-releasing hormone (TRH) is an important extracellular signal substance that acts as a hypothalamic-releasing factor, which stimulates the release of adenohypophyseal hormones and functions as a neurotransmitter/neuromodulator in the central and peripheral nervous system. The inactivation of TRH after its release is catalyzed by an ectoenzyme localized preferentially on neuronal cells in the brain and on lactotrophic pituitary cells. This enzyme exhibits a very high degree of substrate specificity as well as other unusual properties. The activity of the adenohypophyseal enzyme is stringently controlled by estradiol and thyroid hormones, indicating that this enzyme itself may serve regulatory functions. Fragments of the enzyme isolated from rat or pig brain were generated by enzymatic digestion or cyanogen bromide cleavage, purified by reverse-phase HPLC, and sequenced. PCR amplification and screening of cDNA libraries from rat brain and pituitary led to the identification and isolation of a cDNA that encodes a protein of 1025 amino acids. The analysis of the deduced amino acid sequence was consistent with the identification of the enzyme as a glycosylated, membrane-anchored Zn metallopeptidase. Furthermore, Northern blot analysis demonstrated that the mRNA levels paralleled the tissue distribution of the enzyme and that in pituitary tissue the transcript levels rapidly increased when the animals were treated with triiodothyronine. Finally, transient transfection of COS-7 cells with this cDNA led to the expression of an active ectopeptidase that displayed the characteristics of the TRH-degrading ectoenzyme. Images

Schauder, B; Schomburg, L; Kohrle, J; Bauer, K

1994-01-01

178

A conditional suicide system in Escherichia coli based on the intracellular degradation of DNA.  

PubMed Central

The potential risks associated with the intentional or unintentional release of genetically engineered microorganisms led to the construction of biological containment systems by which bacteria are killed in a controlled suicide process. In previously published suicide systems, cell killing was caused by proteins destroying the cell membrane or cell wall. Here a conditional cell killing system based on the intracellular degradation of cellular DNA is presented. The nuclease gene used was that of the extracellular nuclease of Serratia marcescens. The nuclease gene was deleted for the leader-coding sequence, and the truncated gene was put under the control of the lambda pL promoter. Following thermoinduction of the nuclease gene cassette in Escherichia coli, cell survival dropped to 2 x 10(-5), and more than 80% of the radioactively labeled DNA was converted to acid-soluble material within 2.5 h in the absence of cell lysis. The majority (84%) of clones which survived thermoinduced killing turned out to be as sensitive to a second thermoinduction as the original strain. The other clones showed somewhat slower killing kinetics or slightly higher final levels of survivors. The suicide system described combines the regulated killing of cells with the destruction of intracellular DNA otherwise potentially available for horizontal gene transfer processes.

Ahrenholtz, I; Lorenz, M G; Wackernagel, W

1994-01-01

179

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

2013-01-04

180

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

PubMed Central

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.

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

2013-01-01

181

In vitro detection of specific mRNA protection or degradation factors using a fluorescence DNA sequencer  

Microsoft Academic Search

A method to detect specific mRNA degradation factors using non-radioactive RNA synthesis has been developed which involves synthesis of RNA by in vitro transcription in the presence of Texas-Red labeled nucleotides. Degradation of RNA synthesized from the chicken aA-globin 3'-UTR region was detected using an automatic fluorescence DNA sequencer. This fluorescence detection method has higher resolution than the conventional slab

Yukinori Eguchi; Tomoko Eguchi

2002-01-01

182

Laser microdissection and pressure catapulting with PALM® to assist typing of target DNA in dirt samples  

Microsoft Academic Search

Retrieval of genetic profiles from human biological samples mixed with dirt can be difficult using standard DNA extraction methods. Isolation of target cells from debris, using laser microdissection and pressure catapulting prior to DNA extraction, can improve the recovery of genetic profiles of the target component from such samples.

B. L. Lambie-Anoruo; D. V. Prince; I. Koukoulas; D. W. Howells; R. J. Mitchell; R. A. H. van Oorschot

2006-01-01

183

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

PubMed

Testing for mutations in the KRAS oncogene for patients with metastatic colorectal cancer (mCRC) is generally performed using DNA from formalin-fixed paraffin-embedded tumor tissue; however, access to specimens can be limited and analysis challenging. This study assessed the identification of KRAS mutations in circulating free DNA (cfDNA) using a commercially available KRAS polymerase chain reaction (PCR) kit. Matched plasma, serum and tumor samples were available from 71 patients with mCRC who had received prior therapy but whose disease progressed following therapy. Yields of cfDNA from plasma and serum samples were comparable. Analyses were successful in 70/71 plasma-extracted samples (specificity: 97%, sensitivity: 31%) and 67/71 serum- extracted samples (specificity: 100%, sensitivity: 25%). This study demonstrates that KRAS mutations can be detected in cfDNA using a commercially available KRAS PCR kit, confirming cfDNA as a potential alternative source of tumor DNA in a diagnostic setting if access to archival tumor specimens is limited. PMID:22661904

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

2012-05-15

184

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

PubMed Central

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

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

2012-01-01

185

Improved pulsed-field gel electrophoresis procedure for the analysis of Flavobacterium columnare isolates previously affected by DNA degradation.  

PubMed

Flavobacterium columnare is a fresh water bacterium that causes columnaris diseases in over 36 fish species. Intra-species typing of F. columnare can be performed by pulsed-field gel electrophoresis (PFGE). However, this method is hampered by the degradation of chromosomal DNA in about 10% of strains. In the current study, DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. The results substantiate that after problems due to DNases are overcome, PFGE analysis is a reproducible highly discriminating epidemiological method for studying F. columnare isolates regardless of fish host. PMID:18023300

Soto, Esteban; Mauel, Michael; Lawrence, Mark

2007-10-10

186

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

187

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

188

A RAPID METHOD FOR DETECTION AND QUANTIFICATION OF BACTERIAL DNA IN RUST FUNGAL DNA SAMPLES  

Technology Transfer Automated Retrieval System (TEKTRAN)

Rust fungi are obligate parasitic plant pathogens and molecular analysis often depends on extraction of DNA from asexual urediniospores collected from plant tissue. Bacterial DNA contamination of the rust fungal DNA can be a significant problem. A quantitative real-time polymerase chain reaction (Q-...

189

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

SciTech Connect

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

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

1987-05-01

190

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

191

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

PubMed

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

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

2008-08-01

192

Inhibition of gamma-Ray-induced Degradation of E. coli Bs1 DNA by Infection with T1, T2 and T4 Bacteriophage  

Microsoft Academic Search

THE breakdown of DNA in bacteria in response to ionizing radiation has received much attention1-7. This breakdown is mediated by an enzyme or system of enzymes which has not yet been fully characterized. We have investigated the effect of DNA degradation on parental DNA of T1 bacteriophage within the infected cell in radiation conditions which usually break down host DNA.

J. D. Chapman; J. Swez; E. C. Pollard

1968-01-01

193

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

194

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

PubMed

The fingernail hyponychium is an isolated area where biological material may accumulate and can provide a valuable source of evidential material in police investigations. DNA transfer between the victim and suspect frequently occurs during violent crimes and in court there is often reasonable doubt that a mixed DNA profile in a fingernail sample has originated from the assault as the profile may be attributed to previous contact between the two individuals. The purpose of this study was to assess background levels of foreign DNA under the fingernails of individuals from the general population in order to provide data that may help to determine whether DNA transfer occurred during or prior to the assault. Fingernail swabs sampled from 100 volunteers were processed by Qiagen extraction and amplified using AMPFlSTR SGM Plus to obtain DNA profiles. Foreign DNA was detected in 13% of samples, with only 6% of these giving reportable mixed DNA profiles, suggesting the incidence of foreign DNA under the fingernails was low. A significant proportion of the mixed DNA profiles came from male donors; the majority had experienced physical contact within the 24h time period prior to sampling. PMID:19083729

Cook, Olivia; Dixon, Lindsey

2006-11-28

195

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

Microsoft Academic Search

Potent PCR inhibitors in blood and soil samples can cause false negative results from PCR-based clini- cal and forensic tests. We show that the effect of these inhibitors is primarily upon Taq DNA polymer- ase, since mutational alteration of the polymerase can overcome the inhibition to the extent that no DNA purification is now required. An N-terminal deletion (Klentaq1) is

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

2009-01-01

196

Y-STR analysis on DNA mixture samples—Results of a collaborative project of the ENFSI DNA Working Group  

Microsoft Academic Search

The ENFSI (European Network of Forensic Science Institutes) DNA Working Group undertook a collaborative project on Y-STR typing of DNA mixture samples that were centrally prepared and thoroughly tested prior to the shipment. Four commercial Y-STR typing kits (Y-Filer, Applied Biosystems, Foster City, CA, USA; Argus Y Nonaplex, Biotype, Dresden, Germany; Powerplex Y, Promega, Madison, WI, USA; and DYSplex-3, SERAC,

Walther Parson; Harald Niederstätter; Alexandra Lindinger; Peter Gill

2008-01-01

197

Automated Extraction of DNA from Forensic Sample Types Using the PrepFiler Automated Forensic DNA Extraction Kit  

Microsoft Academic Search

The HID EVOlution—Extraction System (Tecan Group Ltd., Mannedorf, Switzerland) was developed to automate DNA extraction from biological samples using the PrepFiler Automated Forensic DNA Extraction Kit (Applied Biosystems, Foster City, CA). The system consists of a Tecan Freedom EVO 150 robot (Tecan Group Ltd., Mannedorf, Switzerland), a graphical user interface designed for use with Freedom EVOware software v 2.1 SPI

Maxim Brevnov; Janna Mundt; Jacki Benfield; Lynda Treat-Clemons; Geert Kalusche; Jason Meredith; Gregory Porter; Manohar R. Furtado; Jaiprakash G. Shewale

2009-01-01

198

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

Microsoft Academic Search

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

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

1997-01-01

199

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.

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

2003-01-01

200

DNA damage in preserved specimens and tissue samples: a molecular assessment.  

PubMed

The extraction of genetic information from preserved tissue samples or museum specimens is a fundamental component of many fields of research, including the Barcode of Life initiative, forensic investigations, biological studies using scat sample analysis, and cancer research utilizing formaldehyde-fixed, paraffin-embedded tissue. Efforts to obtain genetic information from these sources are often hampered by an inability to amplify the desired DNA as a consequence of DNA damage.Previous studies have described techniques for improved DNA extraction from such samples or focused on the effect of damaging agents - such as light, oxygen or formaldehyde - on free nucleotides.We present ongoing work to characterize lesions in DNA samples extracted from preserved specimens. The extracted DNA is digested to single nucleosides with a combination of DNase I, Snake Venom Phosphodiesterase, and Antarctic Phosphatase and then analyzed by HPLC-ESI-TOF-MS.We present data for moth specimens that were preserved dried and pinned with no additional preservative and for frog tissue samples that were preserved in either ethanol, or formaldehyde, or fixed in formaldehyde and then preserved in ethanol. These preservation methods represent the most common methods of preserving animal specimens in museum collections. We observe changes in the nucleoside content of these samples over time, especially a loss of deoxyguanosine. We characterize the fragmentation state of the DNA and aim to identify abundant nucleoside lesions. Finally, simple models are introduced to describe the DNA fragmentation based on nicks and double-strand breaks. PMID:18947416

Zimmermann, Juergen; Hajibabaei, Mehrdad; Blackburn, David C; Hanken, James; Cantin, Elizabeth; Posfai, Janos; Evans, Thomas C

2008-10-23

201

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

PubMed

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. PMID:21703312

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

2011-06-13

202

Quantitative Field Testing Rotylenchulus reniformis DNA from Metagenomic Samples Isolated Directly from Soil  

Microsoft Academic Search

A quantitative PCR procedure targeting the ?-tubulin gene determined the number of Rotylenchulus reniformis Linford & Oliveira 1940 in metagenomic DNA samples isolated from soil. Of note, this outcome was in the presence of other soil-dwelling plant parasitic nematodes including its sister genus Helicotylenchus Steiner, 1945. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated

Kurt Showmaker; Gary W. Lawrence; Shien Lu; Clarissa Balbalian; Vincent P. Klink

2011-01-01

203

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

204

Quantitative Field Testing Rotylenchulus reniformis DNA from Metagenomic Samples Isolated Directly from Soil  

PubMed Central

A quantitative PCR procedure targeting the ?-tubulin gene determined the number of Rotylenchulus reniformis Linford & Oliveira 1940 in metagenomic DNA samples isolated from soil. Of note, this outcome was in the presence of other soil-dwelling plant parasitic nematodes including its sister genus Helicotylenchus Steiner, 1945. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from soil.

Showmaker, Kurt; Lawrence, Gary W.; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P.

2011-01-01

205

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

Microsoft Academic Search

A wide range of methods are commonly used for preserving environmental samples prior to molecular analyses. However, the effect of these preservation methods on fungal DNA is not understood. The objective of this study was to test the effect of eight different preservation methods on the quality and yield of DNA extracted from Bromus inermis and Daucus carota roots colonized

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

2010-01-01

206

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

PubMed Central

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, sediment, 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 target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO4, 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of ?100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10?21 M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.

Chandler, Darrell P.; Stults, Jennie R.; Cebula, Sharon; Schuck, Beatrice L.; Weaver, Derek W.; Anderson, Kevin K.; Egholm, Michael; Brockman, Fred J.

2000-01-01

207

Estimating the effect of SNP genotype on quantitative traits from pooled DNA samples  

PubMed Central

Background Studies to detect associations between DNA markers and traits of interest in humans and livestock benefit from increasing the number of individuals genotyped. Performing association studies on pooled DNA samples can provide greater power for a given cost. For quantitative traits, the effect of an SNP is measured in the units of the trait and here we propose and demonstrate a method to estimate SNP effects on quantitative traits from pooled DNA data. Methods To obtain estimates of SNP effects from pooled DNA samples, we used logistic regression of estimated allele frequencies in pools on phenotype. The method was tested on a simulated dataset, and a beef cattle dataset using a model that included principal components from a genomic correlation matrix derived from the allele frequencies estimated from the pooled samples. The performance of the obtained estimates was evaluated by comparison with estimates obtained using regression of phenotype on genotype from individual samples of DNA. Results For the simulated data, the estimates of SNP effects from pooled DNA are similar but asymptotically different to those from individual DNA data. Error in estimating allele frequencies had a large effect on the accuracy of estimated SNP effects. For the beef cattle dataset, the principal components of the genomic correlation matrix from pooled DNA were consistent with known breed groups, and could be used to account for population stratification. Correctly modeling the contemporary group structure was essential to achieve estimates similar to those from individual DNA data, and pooling DNA from individuals within groups was superior to pooling DNA across groups. For a fixed number of assays, pooled DNA samples produced results that were more correlated with results from individual genotyping data than were results from one random individual assayed from each pool. Conclusions Use of logistic regression of allele frequency on phenotype makes it possible to estimate SNP effects on quantitative traits from pooled DNA samples. With pooled DNA samples, genotyping costs are reduced, and in cases where trait records are abundant this approach is promising to obtain SNP associations for marker-assisted selection.

2012-01-01

208

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

Microsoft Academic Search

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

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

2002-01-01

209

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

210

A non-organic and non-enzymatic extraction method gives higher yields of genomic DNA from whole-blood samples than do nine other methods tested.  

PubMed

We compared ten methods for extraction of DNA from whole blood. Nine methods require incubation with either enzymes or treatment of organic solvents or both. The 'Rapid Method' (RM) (Method 10) avoids the use of organic solvents (phenol/chloroform) and eliminates completely the use of proteinase K. Thus, the time and cost of DNA extraction are reduced significantly. This is accomplished by salting out and precipitation of the cellular proteins in saturated sodium chloride. This method takes less than an hour to completion, without compromising the yield or the quality of DNA. Using RM, we can make DNA from 0.1 ml of whole blood and as little as 0.5 ml of blood yields DNA sufficient to run a few Southern blots. The RM can also be applied to packed cells. The DNA is free of RNA, protein and degrading enzymes. The uncut DNA runs as a typical slow-migrating, high-molecular-weight and undegraded species in an agarose gel. The DNA is suitable for digestion by various restriction endonucleases. This procedure works equally well with fresh blood samples and with those that are stored at 4 degrees C and -70 degrees C. To our knowledge the RM reported here is the safest, fastest and most quantitative and economical method for preparation of DNA from whole blood and cells. PMID:1494032

Lahiri, D K; Bye, S; Nurnberger, J I; Hodes, M E; Crisp, M

1992-12-01

211

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

PubMed Central

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

Wilson, John James

2011-01-01

212

A comparison between direct PCR and extraction to generate DNA profiles from samples retrieved from various substrates.  

PubMed

Direct PCR generates DNA profiles from samples without using the extraction process. During sample extraction, DNA may be lost due to the methods used, which can affect the quality of the DNA profile obtained. This is not the case with direct PCR, where the sample is transferred directly into the PCR tube. Here, we report on the ability of direct PCR to generate DNA profiles from low amounts of control DNA retrieved from various surfaces using PowerPlex 16 HS. A comparison is made with samples undergoing a preliminary extraction stage using QiaAmp DNA Micro kits. Samples subjected to direct PCR generated DNA profiles with higher peak heights and lower allele dropout on all the different substrates tested when compared to the samples subjected to extraction. The amount of DNA retrieved from each substrate also varied even though the same amount of starting material was deposited, proving that the type of substrate can affect the retrieval of DNA. PMID:21925992

Swaran, Yuvaneswari Chandramoulee; Welch, Lindsey

2011-09-16

213

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

SciTech Connect

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

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

1980-01-01

214

Study of a novel sample injection method (floating electrokinetic supercharging) for high-performance microchip electrophoresis of DNA fragments.  

PubMed

Aiming to achieve high-performance analysis of DNA fragments using microchip electrophoresis, we developed a novel sample injection method, which was given the name of floating electrokinetic supercharging (FEKS). In the method, electrokinetic injection (EKI) and ITP preconcentration of samples was performed in a separation channel, connecting two reservoir ports (P3 and P4) on a cross-geometry microchip. At these two stages, side channels, crossing the separation channel, and their ports (P1 and P2) were electrically floated. After the ITP-stacked zones passed the cross-part, they were eluted for detection by using leading ions from P1 and P2 that enabled electrophoresis mode changing rapidly from ITP to zone electrophoresis (ZE). Possible sample leakage at the cross-part toward P1 and P2 was studied in detail on the basis of computer simulation using a CFD-ACE+ software and real experiments, through which it was validated that the analyte recovery to the separation channel was almost complete. The FEKS method successfully contributed to higher resolution and shorter analysis time of DNA fragments on the cross-microchip owing to more rapid switching from ITP status to ZE separation in comparison with our previous EKS procedure realized on a single-channel microchip. Without any degradation of resolution, the achieved LODs were on average ten times better than using conventional pinched injection. PMID:18393341

Hirokawa, Takeshi; Takayama, Yoichi; Arai, Akihiro; Xu, Zhongqi

2008-05-01

215

Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage.  

PubMed

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A and contribute to delaying cell-cycle progression, thereby preventing genomic instability. Here we report that beta-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of beta-TrCP1 and beta-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that beta-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of beta-TrCP expression results in radioresistant DNA synthesis in response to DNA damage--a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that beta-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation. PMID:14603323

Busino, Luca; Donzelli, Maddalena; Chiesa, Massimo; Guardavaccaro, Daniele; Ganoth, Dvora; Dorrello, N Valerio; Hershko, Avram; Pagano, Michele; Draetta, Giulio F

2003-11-01

216

Iron gall ink-induced corrosion of cellulose: aging, degradation and stabilization. Part 2: application on historic sample material  

Microsoft Academic Search

Degradation of cellulose in historic paper by iron gall ink is a synergistic process of both, acid hydrolysis caused by acidic\\u000a ink ingredients and oxidation catalyzed by free iron and\\/or copper ions. The interplay of both reactions was studied according\\u000a to the CCOA method on historic paper samples. Only minute amounts (few mg) of the samples were required to obtain

Ute Henniges; Rebecca Reibke; Gerhard Banik; Enke Huhsmann; Ulrike Hähner; Thomas Prohaska; Antje Potthast

2008-01-01

217

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

PubMed Central

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

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

2008-01-01

218

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

PubMed Central

Background Exploitation of DNA-based analyses of microbial pathogens, and especially simultaneous typing of several virulence-related genes in bacteria is becoming an important objective of public health these days. Results A procedure for sample processing for a confirmative analysis of enterohemorrhagic Escherichia coli (EHEC) on a single colony with DNA chip array was developed and is reported here. The protocol includes application of fragmented genomic DNA from ultrasonicated colonies. The sample processing comprises first 2.5 min of ultrasonic treatment, DNA extraction (2×), and afterwards additional 5 min ultrasonication. Thus, the total sample preparation time for a confirmative analysis of EHEC is nearly 10 min. Additionally, bioinformatic revisions were performed in order to design PCR primers and array probes specific to most conservative regions of the EHEC-associated genes. Six strains with distinct pathogenic properties were selected for this study. At last, the EHEC chip array for a parallel and simultaneous detection of genes etpC-stx1-stx2-eae was designed and examined. This should permit to sense all currently accessible variants of the selected sequences in EHEC types and subtypes. Conclusion In order to implement the DNA chip array-based analysis for direct EHEC detection the sample processing was established in course of this work. However, this sample preparation mode may also be applied to other types of EHEC DNA-based sensing systems.

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

2008-01-01

219

Dual mode polyspermine with tunable degradability for plasmid DNA and siRNA delivery  

Microsoft Academic Search

Stimuli-responsive degradability is an indispensable design component for polymeric gene carriers. In order to obtain enhanced, non-cytotoxic, and molecularly tunable nonviral gene delivery, spermine, a bioavailable small cationic molecule, was polymerized with diacrylate cross-linkers with or without acid-degradable ketal linkages for controlled dual mode-degradability (i.e., differential degradations in the endosome and the cytosol). The effects of ketal to ester ratios

Min Suk Shim; Young Jik Kwon

2011-01-01

220

Degradation of Polar Organic Micropollutants during Riverbank Filtration: Complementary Results from Spatiotemporal Sampling and Push-Pull Tests.  

PubMed

The fate of polar organic micropollutants (logDOW (pH 7) between -4.2 and +3.5) during riverbank filtration (RBF) at the river Thur was studied using both spatiotemporally resolved sampling and single-well push-pull tests (PPT), followed by LC-MS/MS analysis. The Thur is a dynamic prealpine river with an alluvial sandy-gravel aquifer, which is characterized by short groundwater travel times (a few days) from surface water infiltration to groundwater extraction. The spatiotemporal sampling allowed tracing concentration dynamics in the river and the groundwater and revealed persistence for the drug carbamazepine, while the herbicide MCPA (2-methyl-4-chloro-phenoxyacetic acid) and the drug 4-acetamidoantipyrine were very quickly degraded under the prevalent aerobic conditions. The corrosion inhibitor 1H-benzotriazole was degraded slightly, particularly in a transect influenced by river restoration measures. For the first time in situ first-order degradation rate constants for three pesticides and two pharmaceuticals were determined by PPTs, which confirmed the results of the spatiotemporal sampling. Atenolol was transformed almost completely to atenolol acid. Rate constants of 0.1-1.3 h(-1) for MCPA, 2,4-D, mecoprop, atenolol, and diclofenac, corresponding to half-lives of 0.6-6.3 h, demonstrated the great potential of RBF systems to degrade organic micropollutants and simultaneously the applicability of PPTs for micropollutants in such dynamic systems. PMID:24033151

Huntscha, Sebastian; Rodriguez Velosa, Diana M; Schroth, Martin H; Hollender, Juliane

2013-10-02

221

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

PubMed Central

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

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

2013-01-01

222

Y-STR analysis on DNA mixture samples--results of a collaborative project of the ENFSI DNA Working Group.  

PubMed

The ENFSI (European Network of Forensic Science Institutes) DNA Working Group undertook a collaborative project on Y-STR typing of DNA mixture samples that were centrally prepared and thoroughly tested prior to the shipment. Four commercial Y-STR typing kits (Y-Filer, Applied Biosystems, Foster City, CA, USA; Argus Y Nonaplex, Biotype, Dresden, Germany; Powerplex Y, Promega, Madison, WI, USA; and DYSplex-3, SERAC, Bad Homburg, Germany) were used for the amplification of the mixture samples. The results of the study showed a striking inter-laboratory difference of kit performance as determined from the peak heights of the obtained Y-STR genotypes. Variation in quantity and quality of the shipped DNA can be excluded as reason for the observed differences because both samples and shipping conditions were found to be reproducible in an earlier study. The results suggest that in some cases a laboratory-specific optimization process is indicated to reach a comparable sensitivity for the analysis of minute amounts of DNA. PMID:19083827

Parson, Walther; Niederstätter, Harald; Lindinger, Alexandra; Gill, Peter

2008-02-04

223

Real-time total integrated scattering measurements on the Mir spacecraft to evaluate sample degradation in space.  

PubMed

An instrument to measure total integrated scattering (TIS) in space was built as part of the Optical Properties Monitor instrument package and flown on the Russian Mir Space Station in a low Earth orbit. TIS at two wavelengths was measured in space at approximately weekly intervals from 29 April to 26 December 1997 and telemetered to Earth during the mission. Of the 20 TIS samples, 13 are described here to illustrate the performance of the TIS instrument. These include ten optical samples and three thermal control samples. Two optical samples and one thermal control sample were severely degraded by atomic oxygen. All samples received a light dusting of particles during the mission and an additional heavier layer after the samples returned to Earth. The initial brassboard instrument and the validation tests of the flight instrument are also described. PMID:18357293

Hadaway, J B; Ahmad, A; Pezzaniti, J L; Chipman, R A; Wilkes, D R; Hummer, L L; Crandall, D G; Bennett, J M

2001-06-01

224

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

PubMed

In the examination of sexual assault cases, DNA typing of vaginal samples mostly occurs after differential DNA extraction. Notwithstanding the differential extraction method, the DNA profiles from the seminal fraction often show the male alleles at low-level in combination with female alleles. This unfavorable ratio male to female DNA is due to a limited amount of sperm cells and an overwhelming quantity of female cells. In this study, we compared standard cotton and nylon flocked swabs for post-coital vaginal sampling. Twelve couples donated 88 vaginal swabs - 44 cotton, 44 nylon flocked - which were taken with a time since intercourse (TSI) up to 84 h. These vaginal swabs were sorted into categories on the basis of the TSI and submitted to (1) microscopic examination for the presence of male cells, (2) presumptive tests for the detection of seminal fluid and (3) DNA typing. Cellular elution was found to be 6-fold more efficient from the nylon flocked swabs. This makes microscopic analysis less time consuming as the higher cell yield and better cell morphology simplify detection of male cells. Both swab types reveal similar results regarding presumptive tests and male DNA typing. Positive presumptive tests (RSID-semen and PSA) were obtained up to 60 h TSI and male autosomal profiles up to 72 h TSI. Interestingly, over 50% of the samples negative for both presumptive tests resulted in informative male STR profiles. After differential extraction, less DNA was left on the nylon flocked swabs and more male DNA was isolated. Our results imply that the use of nylon flocked swabs for vaginal sampling will improve microscopic analysis and DNA typing in the medical forensic investigation of sexual assault cases. PMID:20129470

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

2009-08-14

225

Options available for labelling nucleic acid samples in DNA microarray-based detection methods.  

PubMed

DNA microarrays are considered by many researchers to be the platform of choice for the high-throughput analysis of nucleic acids. Since the past two decades, they have been used constantly as powerful tools in differential gene expression, SNP genotyping, DNA sequencing, gene discovery, disease diagnostic and pathways reconstruction. Several methods have been developed to enable samples of limited amounts of RNA to be quantified. Here we evaluate classical and up-to-date assays made available for labelling those samples. This review also sheds light on the recently developed strategies that ensure high sensitivity such as sample and signal amplification, quantum dot, surface plasmom resonance, nanoparticles and cationinc polythiophenes. PMID:22510454

Gibriel, Abdullah A Y

2012-04-17

226

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.

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

2013-01-01

227

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

PubMed Central

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

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

2012-01-01

228

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

PubMed

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

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

2012-02-24

229

An improved DNA isolation technique for PCR detection of Strongyloides stercoralis in stool samples.  

PubMed

Strongyloides stercoralis is a nematode that causes severe infections in immunocompromised patients. The low parasitic burden of chronically infected patients makes diagnosis difficult to achieve by conventional methods. Here, an in-house (IH) method for the isolation of parasite DNA from stools and a PCR assay for the molecular diagnosis of S. stercoralis were optimized. DNA yield and purity improved with the IH method which included a step of incubation of stool samples with a glycine-SDS buffer and mechanical disruption prior to DNA extraction. For the PCR assay, the addition of bovine serum albumin was required to neutralize inhibitors present in stool. The analytical sensitivity of the PCR using DNA as template, isolated with the IH method, was superior to the commercial one. This study demonstrates that a combined method that adds the step of glycine-SDS buffer incubation plus mechanical disruption prior to DNA isolation with the commercial kit increased PCR sensitivity to levels of the IH method. Finally, our assay was tested on 17 clinical samples. With the IH method for DNA isolation, a S. stercoralis specific band was detected by PCR in the first stool sample in all patients (17/17), while with the commercial kit, our S. stercoralis-specific band was only observed in 7 samples. The superior efficiency of the IH and combined methods over the commercial kit was demonstrated when applied to clinical samples with low parasitic burden. These results show that the DNA extraction procedure is a key to increase sensitivity of the S. stercoralis PCR assay in stool samples. The method developed here could help to improve the molecular diagnosis of S. stercoralis. PMID:23416126

Repetto, S A; Alba Soto, C D; Cazorla, S I; Tayeldin, M L; Cuello, S; Lasala, M B; Tekiel, V S; González Cappa, S M

2013-02-12

230

Field testing of collection cards for Cannabis sativa samples with a single hexanucleotide DNA marker.  

PubMed

The validity and feasibility of using DNA collection cards in the field for preservation and analysis of Cannabis sativa genotypes were investigated using a highly specific hexanucleotide marker. Collection cards were submitted to the National Marijuana Initiative, which selectively trained and managed the collection of specific types of samples from a variety of participating agencies. Samples collected at seizure sites included fresh marijuana leaf samples, dried "dispensary" samples, U.S. border seizures, and hashish. Using a standardized PCR kit with custom-labeled oligonucleotide primers specific to marijuana, collection cards produced eight genotypes and 13 different alleles, extremely low baselines, and no cross-reactivity with control plant species. Results were produced from all sample types with the exception of hashish. Plant DNA collection cards represent an easily implementable method for the genetic identification and relatedness of C. sativa street and grow site-seized samples with applications for databasing and market disruption. PMID:21644990

Allgeier, Lindsay; Hemenway, John; Shirley, Nicholas; LaNier, Tommy; Coyle, Heather Miller

2011-06-03

231

Comparison of Commercial DNA Extraction Kits for Extraction of Bacterial Genomic DNA from Whole-Blood Samples  

PubMed Central

The demand for molecular diagnostic tests in medical microbiology has highlighted the need for efficient methods of DNA extraction. In addition, it is preferable for these methods to be automated. An example of such a requirement is for the confirmation of meningococcal disease where rapid, sensitive, and specific procedures are required for public health management purposes. Previous studies have shown that whole blood is the preferred method for the isolation of bacterial DNA in meningococcal disease, and in this study, we compare five commercially available kits for the extraction of bacterial genomic DNA from whole-blood samples. These include kits in a 96-well binding plate, 96-well filter plate, and metallic bead formats. The method for all five kits is described, and the sensitivity, specificity, ease of automation, and overall efficiency are determined.

Smith, K.; Diggle, M. A.; Clarke, S. C.

2003-01-01

232

A simple and efficient method for DNA purification from samples of highly clotted blood.  

PubMed

Rapid purification of DNA from samples of highly clotted blood is a challenging problem due to the difficulty in recovering and dispersing blood clots. We developed a new method for discarding the serum-separator gel and rapidly dispersing the blood clots. A special disposable tip was inserted into the serum-separator gel so that the serum-separator gel could be discarded. The blood clot obtained was dispersed into small pieces through a copper mesh (pore size, 250 ?m) in a special dispersing instrument by centrifugation. After lysis of red blood cells and white blood cells, genomic DNA was concentrated and desalted by isopropanol precipitation. The mean yield of DNA purified from a 0.3-ml blood clot was 22.70 ?g in 173 samples of clotted blood cryopreserved for 1 month, and 19.02 ?g in 1,372 samples of clotted blood cryopreserved for >6 months. DNA samples were successfully performed through polymerase chain reaction, real time polymerase chain reaction, and melt curve analysis. Their quality was comparable with that purified directly from EDTA-anticoagulated blood. The new method overcomes the difficulties in recovering and dispersing blood clots, allowing efficient purification of DNA from samples of highly clotted blood. PMID:20549389

Xu, Ruyi; Ye, Ping; Luo, Leiming; Wu, Hongmei; Dong, Jin; Deng, Xinxin

2010-11-01

233

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

PubMed

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 (approximately 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

2009-09-16

234

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.

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

235

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

PubMed Central

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

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

2012-01-01

236

Quantification of the Flavonoid-Degrading Bacterium Eubacterium ramulus in Human Fecal Samples with a Species-Specific Oligonucleotide Hybridization Probe  

PubMed Central

To investigate the occurrence of the flavonoid-degrading bacterium Eubacterium ramulus in the human intestinal tract, an oligonucleotide probe designated S-S-E.ram-0997-a-A-18 was designed and validated, with over 90 bacterial strains representing the dominant described human fecal flora. Application of S-S-E.ram-0997-a-A-18 to fecal samples from 20 subjects indicated the presence of E. ramulus in each individual tested in numbers from 4.4 × 107 to 2.0 × 109 cells/g of fecal dry mass. Six fecal E. ramulus isolates were recognized by S-S-E.ram-0997-a-A-18 but exhibited different band patterns when analyzed by randomly amplified polymorphic DNA.

Simmering, Rainer; Kleessen, Brigitta; Blaut, Michael

1999-01-01

237

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

Microsoft Academic Search

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

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

1995-01-01

238

Rapid Screening of Complex DNA Samples by Single-Molecule Amplification and Sequencing  

Microsoft Academic Search

Microbial cloning makes Sanger sequencing of complex DNA samples possible but is labor intensive. We present a simple, rapid and robust method that enables laboratories without special equipment to perform single-molecule amplicon sequencing, although in a low-throughput manner, from sub-picogram quantities of DNA. The method can also be used for quick quality control of next-generation sequencing libraries, as was demonstrated

Jiaqi Huang; Zongli Zheng; Anders F. Andersson; Lars Engstrand; Weimin Ye

2011-01-01

239

DIRECT BINDING OF GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE TO TELOMERIC DNA PROTECTS TELOMERES AGAINST CHEMOTHERAPY-INDUCED RAPID DEGRADATION  

PubMed Central

GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is a glycolytic enzyme that displays several non-glycolytic activities, including the maintenance and/or protection of telomeres. In this study, we determined the molecular mechanism and biological role of the interaction between GAPDH and human telomeric DNA. Using gel shift assays, we show that recombinant GAPDH binds directly with high affinity (Kd = 45 nM) to a single-stranded oligonucleotide comprising three telomeric DNA repeats and that nucleotides T1, G5 and G6 of the TTAGGG repeat are essential for binding. The stoichiometry of the interaction is 2:1 (DNA: GAPDH), and GAPDH appears to form a high-molecular weight complex when bound to the oligonucleotide. Mutation of Asp32 and Cys149, which are localized to the NAD-binding site and the active site center of GAPDH, respectively, produced mutants that almost completely lost their telomere-binding functions both in vitro and in situ (in A549 human lung cancer cells). Treatment of A549 cells with the chemotherapeutic agents gemcitabine and doxorubicin resulted in increased nuclear localization of expressed wild-type GAPDH, where it protected telomeres against rapid degradation, concomitant with increased resistance to the growth inhibitory effects of these drugs. The non-DNA-binding mutants of GAPDH also localized to the nucleus when expressed in A549 cells, but did not confer any significant protection of telomeres against chemotherapy-induced degradation or growth inhibition, and this occurred without the involvement of caspase activation or apoptosis regulation. Overall, these data demonstrate that GAPDH binds telomeric DNA directly in vitro and may have a biological role in the protection of telomeres against rapid degradation in response to chemotherapeutic agents in A549 human lung cancer cells.

Demarse, Neil A.; Ponnusamy, Suriyan; Spicer, Eleanor K.; Apohan, Elif; Baatz, John E.; Ogretmen, Besim; Davies, Christopher

2009-01-01

240

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

Microsoft Academic Search

Running header: Human Real-Time PCR Quantitation ABSTRACT: Determining the amount of human DNA extracted from a crime scene sample is an important step in DNA profiling. The forensic community relies almost entirely upon a technique (slot blot) to quantitate human DNA that is imprecise, time consuming and labor intensive. We have previously described a method for quantitation of human DNA

Janice A. Nicklas; Eric Buel

241

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

242

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

PubMed

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

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

2013-01-03

243

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

PubMed

In this paper, we report results of a quantitative analysis of the effects of neutrons on DNA, and, specifically, the production of simple and double breaks of plasmid DNA in aqueous solutions with different concentrations of free-radical scavengers. The radiation damage to DNA was evaluated by electrophoresis through agarose gels. The neutron and gamma doses were measured separately with thermoluminescent detectors. In this work, we have also demonstrated usefulness of a new system for positioning and removing samples in channel BH#3 of the IEA-R1 reactor at the Instituto de Pesquisas Energéticas e Nucleares (Brazil) without necessity of interrupting the reactor operation. PMID:21075641

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

2010-10-29

244

Integrated DNA purification, PCR, sample cleanup, and capillary electrophoresis microchip for forensic human identification.  

PubMed

A fully integrated microdevice and process for forensic short tandem repeat (STR) analysis has been developed that includes sequence-specific DNA template purification, polymerase chain reaction (PCR), post-PCR cleanup and inline injection, and capillary electrophoresis (CE). Fragmented genomic DNA is hybridized with biotin-labeled capture oligos and pumped through a fluidized bed of magnetically immobilized streptavidin-coated beads in microchannels where the target DNA is bound to the beads. The bead-DNA conjugates are then transferred into a 250 nL PCR reactor for autosomal STR amplification using one biotin and one fluorescence-labeled primer. The resulting biotin-labeled PCR products are electrophoretically injected through a streptavidin-modified capture gel where they are captured to form a concentrated and purified injection plug. The thermally released sample plug is injected into a 14 cm long CE column for fragment separation and detection. The DNA template capture efficiency provided by the on-chip sequence-specific template purification is determined to be 5.4% using K562 standard DNA. This system can produce full 9-plex STR profiles from 2.5 ng input standard DNA and obtain STR profiles from oral swabs in about 3 hours. This fully integrated microsystem with sample-in-answer-out capability is a significant advance in the development of rapid, sensitive, and reliable micro-total analysis systems for on-site human identification. PMID:21293830

Liu, Peng; Li, Xiujun; Greenspoon, Susan A; Scherer, James R; Mathies, Richard A

2011-02-04

245

Analytical methods for environmental sampling of chemical warfare agents and their degradation products  

SciTech Connect

This first technical conference promoted the standardization of analytical procotols to reliably detect chemical warfare agents and their degradation products in soil, water, and other complex environmental media. This supports the various chemical weapons disposal and emergency preparedness programs, Chemical Weapons Convention treaty compliance, installation restoration and base closure decisions. Five major topics were addressed: Implementation for treaty compliance, installation, restoration and stockpile disposal decisions, existing analytical methods, practical applications of existing analytical techniques, immunoassay technologies, environmental and biological fate of agents and their degradation products. Selected papers have been indexed separately for inclusion in the Energy Science and Technology Database.

Watson, A.P. [ed.] [Oak Ridge National Lab., TN (United States); Kistner, S. [ed.] [Army Center for Health Promotion and Preventive Medicine, Aberdeen Proving Ground, MD (United States)

1995-06-01

246

[A method for preparation of DNA suitable for medico-genetic studies from heparinized blood samples].  

PubMed

Heparin traditionally used as an anticoagulant for blood preparations is one of the most powerful inhibitors of the polymerase chain reaction. The molecular genetic analysis for the purpose of forensic medical expertise encounters difficulties when DNA preparations obtained from heparinized samples have to be used. In our practical work, we were faced with the necessity to use heparin-treated blood samples. It turned out impossible to eliminated heparin from DNA isolated from these samples by the known methods. In order to obviate this difficulty, we have developed a special method for obtaining DNA from heparinized blood preparations suitable for the purpose of forensic medical expertise. The new technique includes the stages of preliminary extraction and purification of the blood cell fraction. PMID:21404528

Ivanov, P L; Leonov, S N

247

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

PubMed

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

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

2009-03-30

248

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

SciTech Connect

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

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

1995-12-31

249

Analytical methods for environmental sampling of chemical warfare agents and their degradation products  

Microsoft Academic Search

This first technical conference promoted the standardization of analytical procotols to reliably detect chemical warfare agents and their degradation products in soil, water, and other complex environmental media. This supports the various chemical weapons disposal and emergency preparedness programs, Chemical Weapons Convention treaty compliance, installation restoration and base closure decisions. Five major topics were addressed: Implementation for treaty compliance, installation,

A. P. Watson; S. Kistner

1995-01-01

250

Degradation of Pseudoviral DNA after Infection of Mouse Cells with Polyoma Pseudovirions*  

PubMed Central

When mouse cells are infected with [3H]-thymidine-labeled polyoma pseudovirions, as much as 11% of the input pseudoviral radioactivity is found in the recipient cell DNA. However, the radioactivity found in the recipient cell DNA virtually disappears when nonradioactive thymidine and deoxycytidine, or D-arabinosyl cytosine, are added to the medium. The radioactivity found in the recipient cell DNA appears to be the result of the breakdown of pseudoviral DNA to precursors of DNA and the utilization of these precursors for the synthesis of cell DNA.

Kashmiri, S. V. S.; Aposhian, H. Vasken

1974-01-01

251

Forensic implications of genetic analyses from degraded DNA—A review  

Microsoft Academic Search

Forensic DNA identification techniques are principally based on determination of the size or sequence of desired PCR products. The fragmentation of DNA templates or the structural modifications that can occur during the decomposition process can impact the outcomes of the analytical procedures. This study reviews the pathways involved in cell death and DNA decomposition and the subsequent difficulties these present

Reza Alaeddini; Simon J. Walsh; Ali Abbas

2010-01-01

252

Effects of oral antioxidant treatment upon the dynamics of human sperm DNA fragmentation and subpopulations of sperm with highly degraded DNA.  

PubMed

The primary aim of this study was to determine the effect of oral antioxidant treatment (1500 mg of l-Carnitine; 60 mg of vitamin C; 20 mg of coenzyme Q10; 10 mg of vitamin E; 10 mg of zinc; 200 ?g of vitamin B9; 50 ?g of selenium; 1 ?g of vitamin B12) during a time period of 3 months upon the dynamics of sperm DNA fragmentation following varying periods of sperm storage (0 h, 2 h, 6 h, 8 h and 24 h) at 37 °C in a cohort of 20 infertile patients diagnosed with asthenoteratozoospermia. A secondary objective was to use the sperm chromatin dispersion test (SCD) to study antioxidant effects upon a specific subpopulation of highly DNA degraded sperm (DDS). Semen parameters and pregnancy rate (PR) were also determined. Results showed a significant improvement of DNA integrity at all incubation points (P < 0.01). The proportion of DDS was also significantly reduced (P < 0.05). Semen analysis data showed a significant increase in concentration, motility, vitality and morphology parameters. Our results suggest that antioxidant treatment improves sperm quality not only in terms of key seminal parameters and basal DNA damage, but also helps to maintain DNA integrity. Prior administration of antioxidants could therefore promote better outcomes following assisted reproductive techniques. PMID:22943406

Abad, C; Amengual, M J; Gosálvez, J; Coward, K; Hannaoui, N; Benet, J; García-Peiró, A; Prats, J

2012-09-03

253

Prevalence and persistence of male DNA identified in mixed saliva samples after intense kissing.  

PubMed

Identification of foreign biological material by genetic profiling is widely used in forensic DNA testing in different cases of sexual violence, sexual abuse or sexual harassment. In all these kinds of sexual assaults, the perpetrator could constrain the victim to kissing. The value of the victim's saliva taken after such an assault has not been investigated in the past with currently widely used molecular methods of extremely high sensitivity (e.g. qPCR) and specificity (e.g. multiplex Y-STR PCR). In our study, 12 voluntary pairs were tested at various intervals after intense kissing and saliva samples were taken from the women to assess the presence of male DNA. Sensitivity-focused assays based on the SRY (single-copy gene) and DYS (multi-copy gene) sequence motifs confirmed the presence of male DNA in female saliva after 10 and even 60min after kissing, respectively. For specificity, standard multiplex Y-STR PCR profiling was performed and male DNA was found in female saliva samples, as the entire Y-STR profile, even after 30min in one sample. Our study confirms that foreign DNA tends to persist for a restricted period of time in the victim's mouth, can be isolated from saliva after prompt collection and can be used as a valuable source of evidence. PMID:22917815

Kamodyová, Natália; Durdiaková, Jaroslava; Celec, Peter; Sedlá?ková, Tatiana; Repiská, Gabriela; Sviežená, Barbara; Minárik, Gabriel

2012-08-20

254

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

PubMed

In recent years, evidence from domestic dogs has increasingly been analyzed by forensic DNA testing. Especially, canine hairs have proved most suitable and practical due to the high rate of hair transfer occurring between dogs and humans. Starting with the description of a contamination-free sample handling procedure, we give a detailed workflow for sequencing hypervariable segments (HVS) of the mtDNA control region from canine evidence. After the hair material is lysed and the DNA extracted by Phenol/Chloroform, the amplification and sequencing strategy comprises the HVS I and II of the canine control region and is optimized for DNA of medium-to-low quality and quantity. The sequencing procedure is based on the Sanger Big-dye deoxy-terminator method and the separation of the sequencing reaction products is performed on a conventional multicolor fluorescence detection capillary electrophoresis platform. Finally, software-aided base calling and sequence interpretation are addressed exemplarily. PMID:22139671

Berger, C; Berger, B; Parson, W

2012-01-01

255

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

PubMed

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

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

2013-07-30

256

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

PubMed

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

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

2012-01-17

257

Purifying and concentrating genomic DNA from mock forensic samples using Millipore Amicon filters.  

PubMed

Regenerated cellulose filters are used for concentrating and purifying genomic DNA from casework samples, due to the high yields and low retentate volumes that these filters provide. The Millipore Ultracel YM-100 is an example of this filter type and has been available to the forensics community for this application since 1990. In 2002, Millipore introduced the Amicon line of vertical filters that provide a larger area for filtration and have a dead space to prevent spinning to dryness. In the present study, Amicon filters were optimized in terms of g force and spin times for their ability to concentrate and purify genomic DNA. The Amicon Ultra 0.5 mL 30 K was used with mock forensic samples containing as little as 160 buccal cells, 20 nL of blood, or 8 nL of semen. In conclusion, the Amicon line of filters can be used to purify genomic DNA from small numbers of cells. PMID:23082821

Garvin, Alex M; Fritsch, Anja

2012-10-19

258

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

PubMed

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

Cox, Annie M; Goodwin, Kelly D

2013-06-20

259

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

PubMed

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

Brudzynski, Katrina; Abubaker, Kamal; Wang, Tony

2012-07-04

260

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

PubMed Central

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

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

1999-01-01

261

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

PubMed Central

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

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

2010-01-01

262

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

PubMed

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

2012-03-14

263

Real-time polymerase chain reaction detection of bovine DNA in meat and bone meal samples.  

PubMed

We describe a real-time polymerase chain reaction (PCR) assay for the detection of bovine DNA extracted from meat and bone meal (MBM) samples. PCR primers were used to amplify a 271-bp region of the mitochondrial ATPase 8-ATPase 6 gene, and a fluorogenic probe (BOV1) labeled with a 5' FAM reporter and a 3' TAMRA quencher was designed to specifically detect bovine PCR product. The specificity of the BOV1 probe for the detection of the bovine PCR product was confirmed by Southern blot hybridization analysis of the probe with PCR products generated from ovine, porcine, and bovine genomic DNA extracted from blood and with PCR products generated from genomic DNA extracted from single-species laboratory scale rendered MBM samples. The specificity of the BOV1 probe was also evaluated in real-time PCR reactions including these genomic targets. Both methods demonstrated that the BOV1 probe was specific for the detection of bovine PCR product. The BOV1 probe had a detection limit of 0.0001% bovine material by Southern blot DNA probe hybridization analysis and a detection limit of 0.001% bovine material in the real-time PCR assay. Application of the real-time PCR assay to six industrial samples that had previously tested positive for the presence of bovine material with a conventional PCR assay yielded positive results with the real-time PCR assay for four samples. PMID:12117251

Lahiff, S; Glennon, M; Lyng, J; Smith, T; Shilton, N; Maher, M

2002-07-01

264

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

Microsoft Academic Search

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

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

1995-01-01

265

Removal of Free Extracellular DNA from Environmental Samples by Ethidium Monoazide and Propidium Monoazide?  

PubMed Central

Recently, new DNA extraction techniques (using ethidium monoazide and propidium monoazide) have been developed to discriminate between alive and dead bacterial cells. Nevertheless, for complex environmental samples, no data are available yet. In the present study, these new methods were applied to anaerobic-fermentor sludge and the results were compared to a conventional microbiological approach.

Wagner, Andreas O.; Malin, Cornelia; Knapp, Brigitte A.; Illmer, Paul

2008-01-01

266

Direct identification of chlamydiae from clinical samples using a DNA microarray assay—A validation study  

Microsoft Academic Search

While DNA microarrays have become a widely accepted tool for mRNA expression monitoring, their use in rapid diagnosis of bacterial and viral pathogens is only emerging. So far, insufficient sensitivity and high costs have been the major limiting factors preventing more widespread use of microarray platforms in direct testing of clinical samples. In the present study, a total of 339

Nicole Borel; Evelyne Kempf; Helmut Hotzel; Evelyn Schubert; Paul Torgerson; Peter Slickers; Ralf Ehricht; Taurai Tasara; Andreas Pospischil; Konrad Sachse

2008-01-01

267

Pneumocystis jirovecii multilocus genotyping in pooled DNA samples: a new approach for clinical and epidemiological studies.  

PubMed

Specific single-nucleotide polymorphisms (SNPs) are recognized as important DNA sequence variations influencing the pathogenesis of Pneumocystis jirovecii and the clinical outcome of Pneumocystis pneumonia, which is a major worldwide cause of illness among immunocompromised patients. Genotyping platforms for pooled DNA samples are promising methodologies for genetic characterization of infectious organisms. We have developed a new typing strategy for P. jirovecii, which consisted of DNA pools prepared according to clinical data (HIV diagnosis, microscopic and molecular detection of P. jirovecii, parasite burden, clinical diagnosis and follow-up of infection) from individual samples using quantitative real-time PCR followed by multiplex-PCR/single base extension (MPCR/SBE). The frequencies of multiple P. jirovecii SNPs (DHFR312, mt85, SOD215 and SOD110) encoded at three distinct loci, the dihydrofolate reductase (DHFR), the mitochondrial large-subunit rRNA (mtLSU rRNA) and the superoxide dismutase (SOD) loci, were estimated in seven DNA pooled samples, representing a total of 100 individual samples. The studied SNPs were confirmed to be associated with distinct clinical parameters of infection such as parasite burden and follow-up. The MPCR/SBE-DNA pooling methodology, described in the present study, was demonstrated to be a useful high-throughput procedure for large-scale P. jirovecii SNPs screening and a powerful tool for evaluation of clinically relevant SNPs potentially related to parasite burden, clinical diagnosis and follow-up of P. jirovecii infection. In further studies, the candidate SNPs mt85, SOD215 and SOD110 may be used as molecular markers in association with MPCR/SBE-DNA pooling to generate useful information for understanding the patterns and causes of Pneumocystis pneumonia. PMID:22487139

Esteves, F; Gaspar, J; de Sousa, B; Antunes, F; Mansinho, K; Matos, O

2012-04-04

268

Soil pesticide residue degradation and soil sample management procedures for environmental forensics  

Microsoft Academic Search

The overall goal of this project was to examine soil sampling procedures of each state regulatory agency that regulates pesticide use in that state, and to offer a standardized soil sampling protocol. ^ A survey of each SLA was conducted to determine the types of containers that are typically used when collecting soil samples for pesticide residue analysis and, on

George N. Saxton

2004-01-01

269

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

PubMed

Microorganisms able to rapidly degrade tetrabromobisphenol A (TBBPA) were domesticated in an anaerobic reactor and added to gradually increased concentrations of TBBPA. After 240 days of domestication, the degradation rate reached 96.0% in cultivated batch experiments lasting 20 days. The optimum cultivating temperature and pH were 30°C and 7.0. The bacterial community's composition and diversity in the reactor was studied by comparative analysis with 16S ribosomal DNA clone libraries. Amplified rDNA restriction analysis of 200 clones from the library indicate that the rDNA richness was high (Coverage C 99.5%) and that evenness was not high (Shannon-Weaver index 2.42). Phylogenetic analysis of 63 bacterial sequences from the reactor libraries demonstrated the presence of Betaproteobacteria (33.1%), Gammaproteobacteria (18.7%), Bacteroidetes (13.9%), Firmicutes (11.4%), Chloroflexi (3.6%), Actinobacteria (0.6%), the candidate division TM7 (4.2%) and other unknown, uncultured bacterial groups (14.5%). Comamonas, Achromobacter, Pseudomonas and Flavobacterium were the dominant types. PMID:22420989

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

2012-02-28

270

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

PubMed

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 Mg(2+)-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

2012-04-19

271

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.

Westra, Edze R.; van Erp, Paul B. G.; Kunne, 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

272

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

PubMed

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

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

2012-08-15

273

Antibiotic Resistance Genes in the Bacteriophage DNA Fraction of Environmental Samples  

PubMed Central

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

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

2011-01-01

274

Patterning of a nanoporous membrane for multi-sample DNA extraction  

NASA Astrophysics Data System (ADS)

A novel method for patterning a nanoporous aluminum oxide membrane was developed using SU-8 to allow for extraction of DNA from multiple samples simultaneously. To facilitate the patterning process, the spin curve for SU-8 2015 on the membrane was determined. The correlation profile of the SU-8 spin curve was similar in form to curves of SU-8 spun on silicon wafers, but with a uniform decrease in thickness across the curve. Characterization of the patterned SU-8 regarding step height and surface roughness was determined using pin-based surface profilometry. The patterned aluminum oxide membrane displayed acceptable flatness and precise pattern replication. Thickness uniformity was also observed to be approximately ±2.8% for a 29.3 µm thick pattern. SEM imaging was used to examine the exposed membrane surface for any visible changes or damage to the membrane caused by the patterning process, with none observed. DNA extraction was performed on the patterned membrane using multiple wells to show the ability of collecting multiple DNA samples simultaneously. DNA, marked with a fluorescent dye, was collected on the membrane and successfully observed using a fluorescence microscope. The techniques developed in this work have application to lab-on-a-chip type systems that include DNA extraction steps.

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

2006-01-01

275

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

PubMed Central

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

Campuzano, Susana; Kuralay, Filiz; Lobo-Castanon, M. Jesus; Bartosik, Martin; Vyavahare, Kedar; Palecek, Emil; Haake, David A.; Wang, Joseph

2011-01-01

276

Ternary monolayers as DNA recognition interfaces for direct and sensitive electrochemical detection in untreated clinical samples.  

PubMed

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

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

2011-02-15

277

Adenovirus E4 34k and E1b 55k Oncoproteins Target Host DNA Ligase IV for Proteasomal Degradation?  

PubMed Central

Cells infected by adenovirus E4 mutants accumulate end-to-end concatemers of the viral genome that are assembled from unit-length viral DNAs by nonhomologous end joining (NHEJ). Genome concatenation can be prevented by expression either of E4 11k (product of E4orf3) or of the complex of E4 34k (product of E4orf6) and E1b 55k. Both E4 11k and the E4 34k/E1b 55k complex prevent concatenation at least in part by inactivation of the host protein Mre11: E4 11k sequesters Mre11 in aggresomes, while the E4 34k/E1b 55k complex participates in a virus-specific E3 ubiquitin ligase that mediates ubiquitination and proteasomal degradation. The E4 34k/E1b 55k complex, but not E4 11k, also inhibits NHEJ activity on internal breaks in the viral genome and on V(D)J recombination substrate plasmids, suggesting that it may interfere with NHEJ independently of its effect on Mre11. We show here that DNA ligase IV, which performs the joining step of NHEJ, is degraded as a consequence of adenovirus infection. Degradation is dependent upon E4 34k and E1b 55k, functional proteasomes, and the activity of cellular cullin 5, a component of the adenoviral ubiquitin ligase. DNA ligase IV also interacts physically with E1b 55k. The data demonstrate that DNA ligase IV, like Mre11, is a substrate for the adenovirus-specific E3 ubiquitin ligase; identify an additional viral approach to prevention of genome concatenation; and provide a mechanism for the general inhibition of NHEJ by adenoviruses.

Baker, Amy; Rohleder, Kent J.; Hanakahi, Les A.; Ketner, Gary

2007-01-01

278

Adenovirus E4 34k and E1b 55k oncoproteins target host DNA ligase IV for proteasomal degradation.  

PubMed

Cells infected by adenovirus E4 mutants accumulate end-to-end concatemers of the viral genome that are assembled from unit-length viral DNAs by nonhomologous end joining (NHEJ). Genome concatenation can be prevented by expression either of E4 11k (product of E4orf3) or of the complex of E4 34k (product of E4orf6) and E1b 55k. Both E4 11k and the E4 34k/E1b 55k complex prevent concatenation at least in part by inactivation of the host protein Mre11: E4 11k sequesters Mre11 in aggresomes, while the E4 34k/E1b 55k complex participates in a virus-specific E3 ubiquitin ligase that mediates ubiquitination and proteasomal degradation. The E4 34k/E1b 55k complex, but not E4 11k, also inhibits NHEJ activity on internal breaks in the viral genome and on V(D)J recombination substrate plasmids, suggesting that it may interfere with NHEJ independently of its effect on Mre11. We show here that DNA ligase IV, which performs the joining step of NHEJ, is degraded as a consequence of adenovirus infection. Degradation is dependent upon E4 34k and E1b 55k, functional proteasomes, and the activity of cellular cullin 5, a component of the adenoviral ubiquitin ligase. DNA ligase IV also interacts physically with E1b 55k. The data demonstrate that DNA ligase IV, like Mre11, is a substrate for the adenovirus-specific E3 ubiquitin ligase; identify an additional viral approach to prevention of genome concatenation; and provide a mechanism for the general inhibition of NHEJ by adenoviruses. PMID:17459921

Baker, Amy; Rohleder, Kent J; Hanakahi, Les A; Ketner, Gary

2007-04-25

279

The first detection of Toxoplasma gondii DNA in environmental fruits and vegetables samples.  

PubMed

Toxoplasma gondii infections are prevalent in humans and animals all over the world. The aim of the study was to estimate the occurrence of T. gondii oocysts in fruits and vegetables and determine the genotype of the parasites. A total number of 216 fruits and vegetables samples were taken from shops and home gardens located in the area of northern Poland. Oocysts were recovered with the flocculation method. Then, real-time polymerase chain reaction (PCR) targeting the B1 gene was used for specific T. gondii detection and quantification. Toxoplasma DNA was found in 21 samples. Genotyping at the SAG2 locus showed SAG2 type I and SAG2 type II. This is the first investigation describing T. gondii DNA identification in a large number of fruits and vegetables samples with rapid molecular detection methods. The results showed that fruits and vegetables contaminated with T. gondii may play a role in the prevalence of toxoplasmosis in Poland. PMID:21948336

Lass, A; Pietkiewicz, H; Szostakowska, B; Myjak, P

2011-09-25

280

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.

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

2011-01-01

281

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

PubMed

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

2011-11-08

282

The legal, social and ethical controversy of the collection and storage of fingerprint profiles and DNA samples in forensic science  

Microsoft Academic Search

The collection and storage of fingerprint profiles and DNA samples in the field of forensic science for nonviolent crimes is highly controversial. While biometric techniques such as fingerprinting have been used in law enforcement since the early 1900s, DNA presents a more invasive and contentious technique as most sampling is of an intimate nature (e.g. buccal swab). A fingerprint is

Katina Michael

2010-01-01

283

Factors affecting the amount of genomic DNA extracted from ape faeces and the identification of an improved sample storage method  

Microsoft Academic Search

Genetic analysis using noninvasively collected samples such as faeces continues to pose a formidable challenge because of unpredictable variation in the extent to which usable DNA is obtained. We investigated the influence of multiple variables on the quantity of DNA extracted from faecal samples from wild mountain gorillas and chimpanzees. There was a small negative correlation between temperature at time

A. M. NSUBUGA; M. M. ROBBINS; A. D. ROEDER; P. A. MORIN; C. BOESCH; L. VIGILANT

2004-01-01

284

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

National Technical Information Service (NTIS)

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

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

2013-01-01

285

A selective requirement for elevated calcium in DNA degradation, but not early events in anti-Fas-induced apoptosis.  

PubMed

Jurkat cells undergo apoptosis in response to anti-Fas antibody through a caspase-dependent death cascade in which calcium signaling has been implicated. We have now evaluated the role of calcium during this death cascade at the single cell level in real time utilizing flow cytometric analysis and confocal microscopy. Fluo-3 and propidium iodide were employed to evaluate calcium fluxes and to discriminate between viable and non-viable cells, respectively. Anti-Fas treatment of Jurkat cells resulted in a sustained increase in intracellular calcium commencing between 1 and 2 h after treatment and persisting until subsequent loss of cell membrane integrity. The significance of this rise in calcium was evaluated by buffering intracellular calcium with BAPTA and/or removing calcium from the extracellular medium and monitoring the effects of these manipulations on calcium signaling and components of the apoptotic process. Complete inhibition of the anti-Fas induced rise in intracellular calcium required both chelation of [Ca(2+)](i) and removal of extracellular calcium. Interestingly, this condition did not abrogate several events in Fas-induced apoptosis including cell shrinkage, mitochondrial depolarization, annexin binding, caspase activation, and nuclear poly(A)DP-ribose polymerase cleavage. Furthermore, calcium-free conditions in the absence of anti-Fas antibody weakly induced these apoptotic components. In marked contrast, calcium depletion did not induce DNA degradation in control cells, and inhibited apoptotic DNA degradation in response to anti-Fas. These data support the concept that the rise in intracellular calcium is not a necessary component for the early signal transduction pathways in anti-Fas-induced apoptosis in Jurkat cells, but rather is necessary for the final degradation of chromatin via nuclease activation. PMID:10859318

Scoltock, A B; Bortner, C D; St J Bird, G; Putney, J W; Cidlowski, J A

2000-09-29

286

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

Microsoft Academic Search

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

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

2002-01-01

287

Heteroplasmic substitutions in the mitochondrial DNA control region in mother and child samples  

Microsoft Academic Search

The sequences of the two hypervariable regions of the mitochondrial DNA control region (HV1 and HV2) from close maternal\\u000a relatives (mother-child pairs) were compared to determine the frequency of mutations between two generations. A total of 68\\u000a blood samples were sequenced only in HV1 and 86 were analysed for HV1 and HV2. The intergenerational comparison led to the\\u000a identification of

J. Hühne; H. Pfeiffer; B. Brinkmann

1998-01-01

288

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

289

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

290

Application of fluorescence in situ hybridization technique to detect simazine-degrading bacteria in soil samples.  

PubMed

We propose a new approach to evaluate the natural attenuation capacity of soil by using fluorescence in situ hybridization (FISH). A specific oligonucleotide probe AtzB1 was designed based on the sequence data of the atzB gene involved in the hydrolytic deamination of s-triazines; this gene, located in a multiple copy plasmid was detected by the optimized FISH protocol. Two agricultural soils (Lodi and Henares) with a history of simazine treatments, and two natural soils (Soto and Monza), without previous exposure to simazine, were studied. AtzB1 probe-target cells were found only in the agricultural soils and, in a greater percentage, in the Lodi soil, compared to the Henares one. Moreover, the greatest percentage of AtzB1 probe-target cells in Lodi was accompanied by a greater mineralization rate, compared to the Henares soil. The FISH method used in this study was suitable for the detection of simazine-degrading bacteria and could be a useful indicator of the potential of soil bioremediation. PMID:18082866

Martín, Margarita; Gibello, Alicia; Lobo, Carmen; Nande, Mar; Garbi, Carlos; Fajardo, Carmen; Barra-Caracciolo, Anna; Grenni, Paola; Martínez-Iñigo, M José

2007-12-20

291

Electrochemical DNA sensor for simultaneous detection of genes encoding two functional enzymes involved in lignin degradation  

Microsoft Academic Search

An electrochemical DNA sensor for simultaneous detection of functional genes encoding manganese peroxidase (MnP) and cellobiose dehydrogenase (CDH) on a gold electrode was developed. After two thiolated capture probes assembled on the electrode surface, the electrode was exposed to a monolayer of 6-mercapto-1-hexanol (MCH) solution to prevent nonspecific adsorption of target DNA and detection probes. Horseradish peroxidase–streptavidin (HRP–SA) conjugate and

Zhen Li; Guangming Zeng; Lin Tang; Yi Zhang; Yuanping Li; Ya Pang; Jie Luo; Yuanyuan Liu

2011-01-01

292

High-Throughput DNA Sample QC Using the Agilent 2200 Tapestation System  

PubMed Central

Rapid adoption of new sequencing technologies means that sample QC in this workflow not only needs to have high throughput capability, but flexibility is also critical. The Agilent 2200 TapeStation system meets this demand by providing a simple to use automated electrophoresis platform with truly variable throughput capabilities, from a single sample up to a 96-well plate. The D1K ScreenTape assay on the 2200 TapeStation enables analysis of DNA fragments between 35 and 1000bp, suiting NGS library preparation QC and quantification. This study is an assessment of the performance the D1K ScreenTape assay in high-throughput DNA analysis using the 96-well sample plate capability. Sizing and quantification were assessed for accuracy and precision on a foil-sealed 96-well plate over typical and extended run times. The study concluded that the D1K ScreenTape assay is suited for accurate sizing and quantification of DNA fragments in high-throughput applications.

Padmanaban, Arunkumar; Inche, Adam; Gassmann, Marcus; Salowsky, Ruediger

2013-01-01

293

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

SciTech Connect

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

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

1981-01-01

294

Evidence for UV-B-induced DNA degradation in Euglena gracilis mediated by activation of metal-dependent nucleases.  

PubMed

It is demonstrated that in vivo irradiation with artificial UV-B for several hours significantly reduces the amount of large DNA extractable from immobilized Euglena in comparison with non-irradiated controls. This UV-B effect can be eliminated by a drastic reduction of the divalent ion concentration in the extracellular medium, i.e. the substitution of the culture medium by Tris-buffered agarose. Moreover, in vitro degradation of large DNA is demonstrated for crude protein extracts isolated from non-irradiated or UV-B-irradiated Euglena. The nuclease activity is shown for both crude protein extracts and purified nucleases; in both cases, two protein bands possessing nuclease activity are obtained with apparent molecular masses of 26 and 40 kDa and their activity is inhibited by specific nuclease inhibitors, i.e. aurintricarboxylic acid and ATP, applied at a concentration as low as 10(-8) M. Moreover, in vitro, nuclease activity clearly depends on the pH, with an optimum around pH 4.5, and on the ion composition of the extracellular medium. A strong stimulating effect is shown for Ca2+ with an optimum around 10(-4) M; this effect is potentiated by Zn2+ and Mn2+, but strongly counteracted by Mg2+ and the calmodulin inhibitors trifluoperazine and N- (6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W5). These results favour the concept which explains the lethal UV-B effect on Euglena as arising from a change in the general metabolic state of the cell and an activation of a DNA-degrading system, i.e. activation of metal-dependent nucleases (U.K. Tirlapur, D.-P. Häder and R. Scheuerlein, UV-B mediated damage in the photosynthetic flagellate, Euglena gracilis, studied by image analysis, Beitr. Biol. Pflanzen, 67 (1992) 305-317). PMID:8583279

Scheuerlein, R; Treml, S; Thar, B; Tirlapur, U K; Häder, D P

1995-12-01

295

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

2010-12-22

296

A simple and effective sample preparation method for atomic force microscopy visualization of individual DNA molecules in situ.  

PubMed

A simple, controllable and effective sample preparation method was established for atomic force microscopy (AFM) imaging of individual DNA molecules in aqueous solution. Firstly, magnesium ion (Mg(2+)) at a concentration of 5.0-10.0 mM as a positively charged bridge was transferred onto mica to immobilize DNA molecules. Then Mg(2+)-modified mica was used to investigate DNA molecules in any buffer without magnesium ion by AFM. AFM images demonstrated that DNA molecules can be successfully observed in solution with good resolution, reproducibility, and stability. Further, this DNA sample preparation method makes AFM successful to investigate DNA molecular interaction in situ and DNA/chitosan complex in gene delivery. PMID:20535564

Shen, Xin-Cheng; Bao, Lei; Zhang, Zhi-Ling; Liu, Xiaoshen; Pang, Dai-Wen; Xu, Jianrong

2010-06-11

297

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

PubMed

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

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

2001-12-18

298

BRCA1 contributes to transcription-coupled repair of DNA damage through polyubiquitination and degradation of Cockayne syndrome B protein.  

PubMed

BRCA1 is an important gene involved in susceptibility to breast and ovarian cancer and its product regulates the cellular response to DNA double-strand breaks. Here, we present evidence that BRCA1 also contributes to the transcription-coupled repair (TCR) of ultraviolet (UV) light-induced DNA damage. BRCA1 immediately accumulates at the sites of UV irradiation-mediated damage in cell nuclei in a manner that is fully dependent on both Cockayne syndrome B (CSB) protein and active transcription. Suppression of BRCA1 expression inhibits the TCR of UV lesions and increases the UV sensitivity of cells proficient in TCR. BRCA1 physically interacts with CSB protein. BRCA1 polyubiquitinates CSB and this polyubiquitination and subsequent degradation of CSB occur following UV irradiation, even in the absence of Cockayne syndrome A (CSA) protein. The depletion of BRCA1 expression increases the UV sensitivity of CSA-deficient cells. These results indicate that BRCA1 is involved in TCR and that a BRCA1-dependent polyubiquitination pathway for CSB exists alongside the CSA-dependent pathway to yield more efficient excision repair of lesions on the transcribed DNA strand. PMID:21756275

Wei, Leizhen; Lan, Li; Yasui, Akira; Tanaka, Kiyoji; Saijo, Masafumi; Matsuzawa, Ayako; Kashiwagi, Risa; Maseki, Emiko; Hu, Yiheng; Parvin, Jeffrey D; Ishioka, Chikashi; Chiba, Natsuko

2011-08-18

299

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

Microsoft Academic Search

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

V. N. Binhi

2007-01-01

300

Spatial scales and probability based sampling in determining levels of benthic community degradation in the Chesapeake Bay.  

PubMed

The extent of degradation of benthic communities of the Chesapeake Bay was determined by applying a previously developed benthic index of biotic integrity at three spatial scales. Allocation of sampling was probability-based allowing areal estimates of degradation with known confidence intervals. The three spatial scales were: (1) the tidal Chesapeake Bay; (2) the Elizabeth River watershed: and (3) two small tidal creeks within the Southern Branch of the Elizabeth River that are part of a sediment contaminant remediation effort. The areas covered varied from 10(-1) to 10(4) km2 and all were sampled in 1999. The Chesapeake Bay was divided into ten strata, the Elizabeth River into five strata and each of the two tidal creeks was a single stratum. The determination of the number and size of strata was based upon consideration of both managerially useful units for restoration and limitations of funding. Within each stratum 25 random locations were sampled for benthic community condition. In 1999 the percent of the benthos with poor benthic community condition for the entire Chesapeake Bay was 47% and varied from 20% at the mouth of the Bay to 72% in the Potomac River. The estimated area of benthos with poor benthic community condition for the Elizabeth River was 64% and varied from 52-92%. Both small tidal creeks had estimates of 76% of poor benthic community condition. These kinds of estimates allow environmental managers to better direct restoration efforts and evaluate progress towards restoration. Patterns of benthic community condition at smaller spatial scales may not be correctly inferred from larger spatial scales. Comparisons of patterns in benthic community condition across spatial scales, and between combinations of strata, must be cautiously interpreted. PMID:12620014

Dauer, Daniel M; Llansó, Roberto J

301

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals  

PubMed Central

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas’ habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

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

2011-01-01

302

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

SciTech Connect

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

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

1986-10-01

303

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

PubMed

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

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

2011-04-01

304

Flow cytometry analysis of changes in the DNA content of the polychlorinated biphenyl degrader Comamonas testosteroni TK102: effect of metabolites on cell-cell separation.  

PubMed

Flow cytometry was used to monitor changes in the DNA content of the polychlorinated biphenyl (PCB)-degrading bacterium Comamonas testosteroni TK102 during growth in the presence or absence of PCBs. In culture medium without PCBs, the majority of stationary-phase cells contained a single chromosome. In the presence of PCBs, the percentage of cells containing two chromosomes increased from 12% to approximately 50%. In contrast, addition of PCBs did not change the DNA contents of three species that are unable to degrade PCBs. In addition, highly chlorinated PCBs that are not degraded by TK102 did not result in a change in the DNA content. These results suggest that PCBs did not affect the DNA content of the cells directly; rather, the intermediate metabolites resulting from the degradation of PCBs caused the increase in DNA content. To study the effect of intermediate metabolites on the DNA content of the cells, four bph genes, bphA1, bphB, bphC, and bphD, were disrupted by gene replacement. The resulting mutant strains accumulated intermediate metabolites when they were grown in the presence of PCBs or biphenyl (BP). When the bphB gene was disrupted, the percentage of cells containing two chromosomes increased in cultures grown with PCBs or BP. When grown with BP, cultures of this mutant accumulated two intermediate metabolites, 2-hydroxybiphenyl (2-OHBP) and 3-OHBP. Addition of 2- or 3-OHBP to a wild-type TK102 and non-PCB-degrading species culture also resulted in an increase in the percentage of cells containing two chromosomes. Electron microscopy revealed that cell-cell separation was inhibited in this culture. This is the first report that hydroxy-BPs can inhibit bacterial cell separation while allowing continued DNA replication. PMID:12324361

Hiraoka, Yoshinori; Yamada, Tohru; Tone, Keiko; Futaesaku, Yutaka; Kimbara, Kazuhide

2002-10-01

305

Reduced sampling efficiency causes degraded Vernier hyperacuity with normal aging: Vernier acuity in position noise  

PubMed Central

Vernier acuity, a form of visual hyperacuity, is amongst the most precise forms of spatial vision. Under optimal conditions Vernier thresholds are much finer than the inter-photoreceptor distance. Achievement of such high precision is based substantially on cortical computations, most likely in the primary visual cortex. Using stimuli with added positional noise, we show that Vernier processing is reduced with advancing age across a wide range of noise levels. Using an ideal observer model, we are able to characterize the mechanisms underlying age-related loss, and show that the reduction in Vernier acuity can be mainly attributed to the reduction in efficiency of sampling, with no significant change in the level of internal position noise, or spatial distortion, in the visual system.

Li, Roger W.; Brown, Brian; Edwards, Marion H.; Ngo, Charlie V.; Chat, Sandy W.; Levi, Dennis M.

2012-01-01

306

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

PubMed Central

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

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

2000-01-01

307

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

2012-02-05

308

Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples  

PubMed Central

Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA.

Whale, Alexandra S.; Cowen, Simon; Foy, Carole A.; Huggett, Jim F.

2013-01-01

309

An Effective Method to Purify Plasmodium falciparum DNA Directly from Clinical Blood Samples for Whole Genome High-Throughput Sequencing  

PubMed Central

Highly parallel sequencing technologies permit cost-effective whole genome sequencing of hundreds of Plasmodium parasites. The ability to sequence clinical Plasmodium samples, extracted directly from patient blood without a culture step, presents a unique opportunity to sample the diversity of “natural” parasite populations in high resolution clinical and epidemiological studies. A major challenge to sequencing clinical Plasmodium samples is the abundance of human DNA, which may substantially reduce the yield of Plasmodium sequence. We tested a range of human white blood cell (WBC) depletion methods on P. falciparum-infected patient samples in search of a method displaying an optimal balance of WBC-removal efficacy, cost, simplicity, and applicability to low resource settings. In the first of a two-part study, combinations of three different WBC depletion methods were tested on 43 patient blood samples in Mali. A two-step combination of Lymphoprep plus Plasmodipur best fitted our requirements, although moderate variability was observed in human DNA quantity. This approach was further assessed in a larger sample of 76 patients from Burkina Faso. WBC-removal efficacy remained high (<30% human DNA in >70% samples) and lower variation was observed in human DNA quantities. In order to assess the Plasmodium sequence yield at different human DNA proportions, 59 samples with up to 60% human DNA contamination were sequenced on the Illumina Genome Analyzer platform. An average ?40-fold coverage of the genome was observed per lane for samples with ?30% human DNA. Even in low resource settings, using a simple two-step combination of Lymphoprep plus Plasmodipur, over 70% of clinical sample preparations should exhibit sufficiently low human DNA quantities to enable ?40-fold sequence coverage of the P. falciparum genome using a single lane on the Illumina Genome Analyzer platform. This approach should greatly facilitate large-scale clinical and epidemiologic studies of P. falciparum.

Clark, Taane G.; Djimde, Abdoulaye A.; Zongo, Issaka; Pinches, Robert; Manske, Magnus; Mangano, Valentina; Alcock, Daniel; Anastasi, Elisa; Maslen, Gareth; MacInnis, Bronwyn; Rockett, Kirk; Modiano, David; Newbold, Christopher I.; Doumbo, Ogobara K.; Ouedraogo, Jean Bosco; Kwiatkowski, Dominic P.

2011-01-01

310

Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded tumor samples.  

PubMed

Analysis of previously unknown genetic aberrations in solid tumors has become possible through the use of comparative genomic hybridization (CGH), which is based on competitive binding of tumor and control DNA to normal metaphase chromosomes. CGH allows detection of DNA sequence copy number changes (deletions, gains, and amplifications) on a genome-wide scale in a single hybridization. We describe here an improved CGH technique, which enables reliable detection of copy number changes in archival formalin-fixed paraffin-embedded tumor samples. The technique includes a modified DNA extraction protocol, which produces high molecular weight DNA which is necessary for high quality CGH. The DNA extraction includes a 3-day digestion with proteinase K, which remarkably improves the yield of high molecular weight DNA. Labeling of the test DNA with a directly fluorescein-conjugated nucleotide (instead of biotin labeling) improved significantly the quality of hybridization. Using the paraffin-block technique, we could analyze 70 to 90% of paraffin blocks, including very old samples as well as samples taken at autopsy. CGH from paraffin blocks was highly concordant (95%) with analyses done from matched freshly frozen tumor samples (n = 5 sample pairs; kappa coefficient = 0.83). The method described here has wide applicability in tumor pathology, allowing large retrospective prognostic studies of genetic aberrations as well as studies on genetic pathogenesis of solid tumors, inasmuch as premalignant lesions and primary and metastatic tumors can be analyzed by using archival paraffin-embedded samples. PMID:7992835

Isola, J; DeVries, S; Chu, L; Ghazvini, S; Waldman, F

1994-12-01

311

Analysis of changes in DNA sequence copy number by comparative genomic hybridization in archival paraffin-embedded tumor samples.  

PubMed Central

Analysis of previously unknown genetic aberrations in solid tumors has become possible through the use of comparative genomic hybridization (CGH), which is based on competitive binding of tumor and control DNA to normal metaphase chromosomes. CGH allows detection of DNA sequence copy number changes (deletions, gains, and amplifications) on a genome-wide scale in a single hybridization. We describe here an improved CGH technique, which enables reliable detection of copy number changes in archival formalin-fixed paraffin-embedded tumor samples. The technique includes a modified DNA extraction protocol, which produces high molecular weight DNA which is necessary for high quality CGH. The DNA extraction includes a 3-day digestion with proteinase K, which remarkably improves the yield of high molecular weight DNA. Labeling of the test DNA with a directly fluorescein-conjugated nucleotide (instead of biotin labeling) improved significantly the quality of hybridization. Using the paraffin-block technique, we could analyze 70 to 90% of paraffin blocks, including very old samples as well as samples taken at autopsy. CGH from paraffin blocks was highly concordant (95%) with analyses done from matched freshly frozen tumor samples (n = 5 sample pairs; kappa coefficient = 0.83). The method described here has wide applicability in tumor pathology, allowing large retrospective prognostic studies of genetic aberrations as well as studies on genetic pathogenesis of solid tumors, inasmuch as premalignant lesions and primary and metastatic tumors can be analyzed by using archival paraffin-embedded samples. Images Figure 1 Figure 3

Isola, J.; DeVries, S.; Chu, L.; Ghazvini, S.; Waldman, F.

1994-01-01

312

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

PubMed

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

Zygner, W; Górski, P; Wedrychowicz, H

2009-04-11

313

A simple and effective sample preparation method for atomic force microscopy visualization of individual DNA molecules in situ  

Microsoft Academic Search

A simple, controllable and effective sample preparation method was established for atomic force microscopy (AFM) imaging of\\u000a individual DNA molecules in aqueous solution. Firstly, magnesium ion (Mg2+) at a concentration of 5.0–10.0 mM as a positively charged bridge was transferred onto mica to immobilize DNA molecules.\\u000a Then Mg2+-modified mica was used to investigate DNA molecules in any buffer without magnesium ion

Xin-Cheng Shen; Lei Bao; Zhi-Ling Zhang; Xiaoshen Liu; Dai-Wen Pang; Jianrong Xu

2011-01-01

314

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

Microsoft Academic Search

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

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

1998-01-01

315

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

Microsoft Academic Search

An important problem in measurement of messenger RNA (mRNA) levels by reverse transcription-polymerase chain reaction (RT-PCR) is DNA contamination, which can produce artifactually increased mRNA concentration. Current methods to eliminate contaminating DNA can compromise the integrity of the RNA, are time-consuming, and\\/or are hazardous. We present a rapid, nuclease-free, and cost-effective method of eliminating contaminating DNA in RNA samples using

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

2009-01-01

316

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.

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

317

Developing equine mtDNA profiling for forensic application  

Microsoft Academic Search

Horse mtDNA profiling can be useful in forensic work investigating degraded samples, hair shafts or highly dilute samples.\\u000a Degraded DNA often does not allow sequencing of fragments longer than 200 nucleotides. In this study we therefore search for\\u000a the most discriminatory sections within the hypervariable horse mtDNA control region. Among a random sample of 39 horses,\\u000a 32 different sequences were

Susan M. R. Gurney; Sandra Schneider; René Pflugradt; Elizabeth Barrett; Anna Catharina Forster; Bernd Brinkmann; Thomas Jansen; Peter Forster

2010-01-01

318

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

SciTech Connect

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

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

2001-12-01

319

Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples  

PubMed Central

Background The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility. Results In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9% (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom® MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total). Conclusions The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving interval plausibly harbouring causative variants contributing to heritable variation. To our knowledge, this is the first report describing sequencing of targeted genomic regions in any livestock species using groups with divergent phenotypes for an economically important trait.

2012-01-01

320

Quantitative Polymerase Chain Reaction with Enzyme-Linked Immunosorbent Assay Detection of Selectively Digested Amplified Sample and Control DNA  

Microsoft Academic Search

A quantitative polymerase chain reaction (PCR) method for the exact quantitation of DNA is described that does not require radioactive labeling or electrophoretic separation of product species, thereby avoiding hazardous and time-consuming procedures which have so far impeded routine use of PCR, in particular in the clinical laboratory. Sample and internal control DNA are competitively amplified in a one-tube nested

M. Hahn; V. Dorsam; P. Friedhoff; A. Fritz; A. Pingoud

1995-01-01

321

Detection and Genotyping of Arcobacter and Campylobacter Isolates from Retail Chicken Samples by Use of DNA Oligonucleotide Arrays  

Microsoft Academic Search

To explore the use of DNA microarrays for pathogen detection in food, we produced DNA oligonucleotide arrays to simultaneously determine the presence of Arcobacter and the presence of Campylobacter in retail chicken samples. Probes were selected that target housekeeping and virulence-associated genes in both Arcobacter butzleri and thermotolerant Campylobacter jejuni and Campylobacter coli. These microarrays showed a high level of

Beatriz Quinones; Craig T. Parker; John M. Janda; W. G. Miller; R. E. Mandrell

2007-01-01

322

Chromatin immunoprecipitation (ChIP): revisiting the efficacy of sample preparation, sonication, quantification of sheared DNA, and analysis via PCR.  

PubMed

The "quantitative" ChIP, a tool commonly used to study protein-DNA interactions in cells and tissue, is a difficult assay often plagued with technical error. We present, herein, the process required to merge multiple protocols into a quick, reliable and easy method and an approach to accurately quantify ChIP DNA prior to performing PCR. We demonstrate that high intensity sonication for at least 30 min is required for full cellular disruption and maximum DNA recovery because ChIP lysis buffers fail to lyse formaldehyde-fixed cells. In addition, extracting ChIP DNA with chelex-100 yields samples that are too dilute for evaluation of shearing efficiency or quantification via nanospectrophotometry. However, DNA extracted from the Mock-ChIP supernatant via the phenol-chloroform-isoamyl alcohol (PCIA) method can be used to evaluate DNA shearing efficiency and used as the standard in a fluorescence-based microplate assay. This enabled accurate quantification of DNA in chelex-extracted ChIP samples and normalization to total DNA concentration prior to performing real-time PCR (rtPCR). Thus, a quick ChIP assay that can be completed in nine bench hours over two days has been validated along with a rapid, accurate and repeatable way to quantify ChIP DNA. The resulting rtPCR data more accurately depicts treatment effects on protein-DNA interactions of interest. PMID:22046253

Schoppee Bortz, Pamela D; Wamhoff, Brian R

2011-10-25

323

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

PubMed Central

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

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

2013-01-01

324

The TREX1 exonuclease R114H mutation in Aicardi-Goutières syndrome and lupus reveals dimeric structure requirements for DNA degradation activity.  

PubMed

Mutations in the TREX1 gene cause Aicardi-Goutières syndrome (AGS) and are linked to the autoimmune disease systemic lupus erythematosus. The TREX1 protein is a dimeric 3' DNA exonuclease that degrades DNA to prevent inappropriate immune activation. One of the most common TREX1 mutations, R114H, causes AGS as a homozygous and compound heterozygous mutation and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1 proteins containing R114H and the insertion mutations aspartate at position 201 (D201ins) and alanine at position 124 (A124ins), found in compound heterozygous AGS with R114H, were prepared and the DNA degradation activities were tested. The homodimer TREX1(R114H/R114H) exhibits a 23-fold reduced single-stranded DNA (ssDNA) exonuclease activity relative to TREX1(WT). The TREX1(D201ins/D201ins) and TREX1(A124ins/A124ins) exhibit more than 10,000-fold reduced ssDNA degradation activities. However, the TREX1(R114H/D201ins) and TREX1(R114H/A124ins) compound heterodimers exhibit activities 10-fold greater than the TREX1(R114H/R114H) homodimer during ssDNA and double-stranded DNA (dsDNA) degradation. These higher levels of activities measured in the TREX1(R114H/D201ins) and TREX1(R114H/A124ins) compound heterodimers are attributed to Arg-114 residues of TREX1(D201ins) and TREX1(A124ins) positioned at the dimer interface contributing to the active sites of the opposing TREX1(R114H) protomer. This interpretation is further supported by exonuclease activities measured for TREX1 enzymes containing R114A and R114K mutations. These biochemical data provide direct evidence for TREX1 residues in one protomer contributing to DNA degradation catalyzed in the opposing protomer and help to explain the dimeric TREX1 structure required for full catalytic competency. PMID:21937424

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

2011-09-21

325

Selective Nucleic Acid Removal via Exclusion (SNARE): Capturing mRNA and DNA from a Single Sample.  

PubMed

The path from gene (DNA) to gene product (RNA or protein) is the foundation of genotype giving rise to phenotype. Comparison of genomic analyses (DNA) with paired transcriptomic studies (mRNA) is critical to evaluating the pathogenic processes that give rise to human disease. The ability to analyze both DNA and mRNA from the same sample is not only important for biologic interrogation but also to minimize variance (e.g., sample loss) unrelated to the biology. Existing methods for RNA and DNA purification from a single sample are typically time-consuming and labor intensive or require large sample sizes to split for separate RNA and DNA extraction procedures. Thus, there is a need for more efficient and cost-effective methods to purify both RNA and DNA from a single sample. To address this need, we have developed a technique, termed SNARE (Selective Nucleic Acid Removal via Exclusion), that uses pinned oil interfaces to simultaneous purify mRNA and DNA from a single sample. A unique advantage of SNARE is the elimination of dilutive wash and centrifugation processes that are fundamental to conventional methods where sample is typically discarded. This minimizes loss and maximizes recovery by allowing nondilutive reinterrogation of the sample. We demonstrate that SNARE is more sensitive than commercially available kits, robustly and repeatably achieving mRNA and DNA purification from extremely low numbers of cells for downstream analyses. In addition to sensitivity, SNARE is fast, easy to use, and cost-effective and requires no laboratory infrastructure or hazardous chemicals. We demonstrate the clinical utility of the SNARE with prostate cancer circulating tumor cells to demonstrate its ability to perform both genomic and transcriptomic interrogation on rare cell populations that would be difficult to achieve with any current method. PMID:24016179

Strotman, Lindsay; O'Connell, Rachel; Casavant, Benjamin P; Berry, Scott M; Sperger, Jamie M; Lang, Joshua M; Beebe, David J

2013-09-26

326

Enhanced Retrieval of DNA from Human Fecal Samples Results in Improved Performance of Colorectal Cancer Screening Test  

PubMed Central

Colorectal cancer accounts for more than 10% of all cancer deaths but is curable, if detected early. We reported previously on a stool-based screening test in which DNA from stool samples is subjected to genome analysis; sensitivity of the test has been limited in part by inefficiency of retrieving DNA from stool. Our aim was to test the impact of a new purification method that would increase the yield of human DNA from stool. DNA from 86 cancer and 100 non-cancer subjects (diagnosed by colonoscopy) were purified from stool with a new method for DNA recovery based on sequence-specific capture with acrylamide gel immobilized capture probes as well as with a previously developed magnetic bead-capture procedure. The new purification method gives an average 5.4-fold increase in the quantity of human DNA that can routinely be retrieved from fecal samples. The increased recovery of DNA corresponds with an increase in assay sensitivity from 53% (CI: 42 to 64%) to 70% (CI: 59 to 79%); P = 0.0005 (by McNemar’s test), with no change in specificity. The newly developed sample preparation method mitigates a major problem in detecting rare cancer-associated genetic changes in heterogeneous clinical samples such as stool.

Whitney, Duncan; Skoletsky, Joel; Moore, Kent; Boynton, Kevin; Kann, Lisa; Brand, Randall; Syngal, Sapna; Lawson, Michael; Shuber, Anthony

2004-01-01

327

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

328

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

PubMed

The FTA technology was applied for sampling, archiving, and molecular analysis of the DNA isolated from stool samples to diagnose and identify microsporidia, the intracellular opportunistic parasites which induce malabsortion syndrome in immunosuppressed humans, particularly in patients with AIDS. Microsporidia DNA was successfully amplified in 6 of 50 stool samples of HIV-positive patients of the S. P. Botkin Memorial Infectious Disease Hospital (St. Petersburg) applied to FTA cards (FTA-Cars, Whatman Inc. Florham Park, NJ, USA). Amplicons (the fragments of rDNA) were directly sequenced, and microsporidia species--Encephalitozoon intestinalis, E. cuniculi, E. hellem, and Enterocytozoon bieneusi--were identified in Genbank by NCBI BLAST program. The FTA method of DNA immobilization is especially promising for epidemiological and field population studies which involve genotyping of microsporidia species and isolates. PMID:22332422

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

2011-01-01

329

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

PubMed Central

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

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

2011-01-01

330

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

PubMed Central

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

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

2012-01-01

331

The impact of data degradation and sample size on the performance of two similarity coefficients used in behavioural linkage analysis.  

PubMed

In order to determine whether a series of unsolved crimes has been committed by the same offender, the police often must rely on an analysis of behavioural evidence. When carrying out this task, some type of similarity coefficient is typically relied on to assess the degree of behavioural stability and distinctiveness that exists across a set of crimes and questions inevitably arise as to which coefficient to use. In cases of juvenile sex offences, research has suggested that a taxonomic similarity index outperforms the most commonly used metric at the moment, Jaccard's coefficient, especially under conditions of data degradation (missing data). However, recent research has failed to replicate this result in cases of serial homicide and burglary, especially when relatively large sample sizes are used. The current study provides further support for these recent findings using adult serial sexual assault data. Across a range of conditions, the current study demonstrates that Jaccard's coefficient slightly outperforms the taxonomic similarity index on a measure of linking accuracy. Potential explanations for the results are provided, implications are discussed, and future research directions are presented. PMID:20456879

Bennell, Craig; Gauthier, Donna; Gauthier, Donald; Melnyk, Tamara; Musolino, Evanya

2010-04-24

332

Genetic Identification of Missing Persons: DNA Analysis of Human Remains and Compromised Samples  

Microsoft Academic Search

Human identification has made great strides over the past 2 decades due to the advent of DNA typing. Forensic DNA typing provides genetic data from a variety of materials and individuals, and is applied to many important issues that confront society. Part of the success of DNA typing is the generation of DNA databases to help identify missing persons and

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

2012-01-01

333

[Consent in DNA sample harvesting. With special reference to processes with minors (Part I)].  

PubMed

At present, there is no doubt as to the enormous importance of genetic testing for DNA markers in investigating crimes and identifying the guilty parties. We consider it unnecessary to sing the virtues of this celebrated method, which is now generally permitted by our courts, and which is forcing us to resolve diverse matters that the scarcity and insufficiency of current legislation is unable to settle. The objective of this paper is to analyse the consent given by the affected party to having genetic samples taken, as well as the inclusion of their profiles into police databases afterwards. This consent appears to be the main source of legitimacy in these scenarios. However, in order for it to take full effect, certain requirements must be met, especially in relation to the affected party giving informed consent. This means that in order for the consent to be considered valid, the victim must be informed of the legal scope and consequences that may arise from the test, as well as the legal consequences that may arise from their refusal. In the case of children, debate is needed as to whether they can be asked to give a genetic sample, if court authorisation or permission from their legal representatives or even their lawyer is always necessary or whether the informed, voluntary consent of the child might be sufficient, also whether police databases can or should have access to these samples. PMID:22977956

de Neyra Kappler, Susana Alvarez

334

DNA-PCR analysis of bloodstains sampled by the polyvinyl-alcohol method.  

PubMed

Among the usual techniques of sampling gunshot residues (GSR), the polyvinyl-alcohol method (PVAL) includes the advantage of embedding all particles, foreign bodies and stains on the surface of the shooter's hand in exact and reproducible topographic localization. The aim of the present study on ten persons killed by firearms was to check the possibility of DNA-PCR typing of blood traces embedded in the PVAL gloves in a second step following GSR analysis. The results of these examinations verify that the PVAL technique does not include factors that inhibit successful PCR typing. Thus the PVAL method can be recommended as a combination technique to secure and preserve inorganic and biological traces at the same time. PMID:9987876

Schyma, C; Huckenbeck, W; Bonte, W

1999-01-01

335

Tagging the Signatures of Domestication in Common Bean (Phaseolus vulgaris) by Means of Pooled DNA Samples  

PubMed Central

Background and Aims The main aim of this study was to use an amplified fragment length polymorphism (AFLP)-based, large-scale screening of the whole genome of Phaseolus vulgaris to determine the effects of selection on the structure of the genetic diversity in wild and domesticated populations. Methods Using pooled DNA samples, seven each of wild and domesticated populations of P. vulgaris were studied using 2506 AFLP markers (on average, one every 250 kb). About 10 % of the markers were also analysed on individual genotypes and were used to infer allelic frequencies empirically from bulk data. In both data sets, tests were made to determine the departure from neutral expectation for each marker using an FST-based method. Key Results The most important outcome is that a large fraction of the genome of the common bean (16 %; P < 0·01) appears to have been subjected to effects of selection during domestication. Markers obtained in individual genotypes were also mapped and classified according to their proximities to known genes and quantitative trait loci (QTLs) of the domestication syndrome. Most of the markers that were found to be potentially under the effects of selection were located in the proximity of previously mapped genes and QTLs related to the domestication syndrome. Conclusions Overall, the results indicate that in P. vulgaris a large portion of the genome appears to have been subjected to the effects of selection, probably because of linkage to the loci selected during domestication. As most of the markers that are under the effects of selection are linked to known loci related to the domestication syndrome, it is concluded that population genomics approaches are very efficient in detecting QTLs. A method based on bulk DNA samples is presented that is effective in pre-screening for a large number of markers to determine selection signatures.

Papa, Roberto; Bellucci, Elisa; Rossi, Monica; Leonardi, Stefano; Rau, Domenico; Gepts, Paul; Nanni, Laura; Attene, Giovanna

2007-01-01

336

A new dielectric response model for water tree degraded XLPE insulation - part a: model development with small sample verification  

Microsoft Academic Search

Water tree degradation in underground XLPE insulated cables is a growing, worldwide problem. This form of degradation is ultimately fatal for affected cables, and therefore the detection of damaging trees in power cable insulation is vital for distribution companies to avoid catastrophic failure. Dielectric response measurements, in both the time and frequency domains, can generate valuable information about the condition

Andrew J. Thomas; Tapan K. Saha

2008-01-01

337

The Achilles tendon as a DNA source for STR typing of highly decayed corpses  

Microsoft Academic Search

Forensic criminal casework often involves DNA profiling of human postmortem tissues, whereas degradational processes can affect PCR-based Short Tandem Repeat (STR) analysis. Degradation of DNA is observed to vary among different tissues and with time. Therefore, the stability of DNA in Achilles tendon samples is compared to that in muscle and kidney specimens with a variety of postmortem histories. Tissue

A. Roeper; W. Reichert; R. Mattern

2007-01-01

338

Abklärung strittiger Identität von Blutalkoholproben mit DNA-Fingerprinting  

Microsoft Academic Search

DNA fingerprinting is a perfect tool for investigating the identity of disputed blood by alcohol samples extracted. However, blood samples stored at an ambient temperature for longer periods can show considerable degradation of high-molecular DNA, diminishing the value of fingerprint investigation because of loss of the less frequent bands formed by the longer DNA fragments. Addition of the complexing agent

W. Bär; A. Kratzer

1989-01-01

339

EFFECT OF DIFFERENT PRESERVATIVES ON THE HUMAN SOFT TISSUES STORED AT DIFFERENT TEMPERATURES AND INTERVALS OF TIME FOR THE ISOLATION OF DNA FOR DNA FINGERPRINTING  

Microsoft Academic Search

The major problem faced by the forensic laboratories is to get the samples in proper conditions. Most of the biological samples brought for DNA analysis are either partially degraded or completely degraded and many a times it is not possible to extract DNA from them. It necessitates the optimization of the temperature conditions and preservatives for the storage of the

Anupuma Raina; Bhuvnesh K. Yadav; S Lalwani; TD Dogra

2006-01-01

340

Antioxidant properties of neohesperidin dihydrochalcone: inhibition of hypochlorous acid-induced DNA strand breakage, protein degradation, and cell death.  

PubMed

Neohesperidin dihydrochalcone (NHDC), a non-nutritive sweetening agent, is simply produced by hydrogenation of neohesperidin. The aim of this study is to evaluate the antioxidant and radical scavenging properties of neohesperidin dihydrochalcone and other structurally related compounds (phloridzin, neohesperidin) toward different reactive radical and oxygen species including .ABTS+, .O2-, .OH, H2O2, and HOCl in vitro. NHDC showed remarkable radical scavenging activity against stable radical and reactive oxygen species (ROS) in concentration dependent manner. Especially, NHDC was the most potent inhibitor of H2O2 and HOCl. NHDC showed HOCl scavenging activity of 93.5% and H2O2 scavenging property of 73.5% which was more than those of all the tested compounds including ascorbic acid and BHT. Moreover, NHDC could inhibit protein degradation, plasmid DNA strand cleavage and HIT-T15, HUVEC cell death from HOCl attack while mannitol, BHT, and ascorbic acid could not protect them effectively. These results suggest that NHDC is a potent antioxidant, especially it is evaluated as a novel HOCl scavenger. This study implies the possibility of therapeutic effect of NHDC on ROS-related inflammatory diseases. PMID:17268074

Choi, Je-Min; Yoon, Byoung-Seok; Lee, Sang-Kyou; Hwang, Jae-Kwan; Ryang, Ryung

2007-02-01

341

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

PubMed Central

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

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

2006-01-01

342

Effects of humic acid and suspended soils on adsorption and photo-degradation of microcystin-LR onto samples from Taiwan reservoirs and rivers.  

PubMed

This article covers the adsorption capacity of microcystin-LR (MC-LR) onto natural organic matter (NOM) or suspended solids of water samples from reservoirs (Emerald and Jade reservoirs) and rivers (Dongshan, Erhjen and Wukai rivers) in Taiwan to determine the fate, transport behavior and photo-degradation of microcystins in natural water systems. Langmuir adsorption and photo-degradation studies were carried out and the capability of samples for MC-LR adsorption was confirmed. Among these, samples collected from reservoir showed enhanced MC-LR adsorption than that of river samples and the greater adsorption behavior was always favored by larger content of organic matter and suspended particles in the system. It is obvious from the experimental results that the adsorption of MC-LR was influenced by suspended particles (turbidity), humic acid (HA), organic matter content and other pollutants. The effective photo-degradation of MC-LR was achieved using higher energy, lower wavelength (254 nm) UV light within 60 min. The presence of humic acid and turbidity affected the photo-degradation process. These data provide important information that may be applied to management strategies for improvement of water quality in reservoirs and rivers and other water bodies in Taiwan. PMID:22476095

Thirumavalavan, Munusamy; Hu, Ya-Lan; Lee, Jiunn-Fwu

2012-03-19

343

Field-amplified sample stacking for the detection of chemical warfare agent degradation products in low-conductivity matrices by capillary electrophoresis-mass spectrometry  

Microsoft Academic Search

Preconcentration of chemical warfare agent degradation products (alkylphosphonic acids and alkyl alkylphosphonic acids) in low-conductivity matrices (purified water, tap water and local river water) by field-amplified sample stacking (FASS) was developed for capillary electrophoresis (CE) coupled to ion trap mass spectrometry. FASS was performed by adding a mixture of HCOONH4 and NH4OH in appropriate concentrations to the sample. This allowed

Mélanie Lagarrigue; Anne Bossée; Arlette Bégos; Nathalie Delaunay; Anne Varenne; Pierre Gareil; Bruno Bellier

2008-01-01

344

Inference from Samples of DNA Sequences Using a Two-Locus Model  

PubMed Central

Abstract Performing inference on contemporary samples of DNA sequence data is an important and challenging task. Computationally intensive methods such as importance sampling (IS) are attractive because they make full use of the available data, but in the presence of recombination the large state space of genealogies can be prohibitive. In this article, we make progress by developing an efficient IS proposal distribution for a two-locus model of sequence data. We show that the proposal developed here leads to much greater efficiency, outperforming existing IS methods that could be adapted to this model. Among several possible applications, the algorithm can be used to find maximum likelihood estimates for mutation and crossover rates, and to perform ancestral inference. We illustrate the method on previously reported sequence data covering two loci either side of the well-studied TAP2 recombination hotspot. The two loci are themselves largely non-recombining, so we obtain a gene tree at each locus and are able to infer in detail the effect of the hotspot on their joint ancestry. We summarize this joint ancestry by introducing the gene graph, a summary of the well-known ancestral recombination graph.

Griffiths, Robert C.

2011-01-01

345

Bacterial diversity in a soil sample from a subtropical Australian environment as determined by 16S rDNA analysis  

Microsoft Academic Search

In order to investigate the genetic diver- sity of streptomycetes in an acid forested soil sample from Mt. Coot-tha, Brisbane, Australia, cells were mechani- cally lysed within the soil matrix and genomic DNA was isolated and purified. 16S ribosomal (r)DNA was am- plified by the polymerase chain reaction (PCR) method using one primer conserved for members of the domain Bacteria

E. STACK; BRANIYr W. LIESACX; B. M. GOEBEL

346

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

347

Southern and dot blot analysis of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas  

Microsoft Academic Search

Summary  Two extraction methods for the isolation of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas\\u000a were compared. The processed DNAs were compared with DNAs from fresh specimens of the same tumors. The two extraction methods\\u000a gave similar results. Formalin-fixation and paraffinembedding irreversibly denatured DNA and consequently decreased the extraction\\u000a yield and interfered with the quantitative measurement of DNA.\\u000a \\u000a Southern

Peter T. Moerkerk; Han J. Kessels; Joop ten Kate; Antony F. P. M. de Goeij; Fré T. Bosman

1989-01-01

348

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

PubMed Central

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

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

2012-01-01

349

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

PubMed

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

Be, Nicholas A; Thissen, James B; Gardner, Shea N; McLoughlin, Kevin S; Fofanov, Viacheslav Y; Koshinsky, Heather; Ellingson, Sally R; Brettin, Thomas S; Jackson, Paul J; Jaing, Crystal J

2013-09-09

350

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

PubMed Central

Two PCR protocols targeting the 18S rRNA gene of Cryptococcus neoformans were established, compared, and evaluated in murine cryptococcal meningitis. One protocol was designed as a nested PCR to be performed in conventional block thermal cyclers. The other protocol was designed as a quantitative single-round PCR adapted to LightCycler technology. One hundred brain homogenates and dilutions originating from 20 ICR mice treated with different azoles were examined. A fungal burden of 3 × 101 to 2.9 × 104 CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity of 1 CFU of C. neoformans per mg of brain tissue and less than 10 CFU per volume used for extraction was observed for both PCR protocols, while homogenates of 70 organs from mice infected with other fungi were PCR negative. Specificity testing was performed with genomic DNA from 31 hymenomycetous fungal species and from the ustilaginomycetous yeast Malassezia furfur, which are phylogenetically related to C. neoformans. Twenty-four strains, including species of human skin flora like M. furfur and Trichosporon spp., were PCR negative. Amplification was observed with Cryptococcus amylolentus, Filobasidiella depauperata, Cryptococcus laurentii, and five species unrelated to clinical specimens. LightCycler PCR products from F. depauperata and Trichosporon faecale could be clearly discriminated by melting curve analysis. The sensitive and specific nested PCR assay as well as the rapid and quantitative LightCycler PCR assay might be useful for the diagnosis and monitoring of human cryptococcal infections.

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

2002-01-01

351

What technique should be used for routine detection and quantification of HBV DNA in clinical samples?  

Microsoft Academic Search

Detection of hepatitis B virus (HBV) DNA in serum allows monitoring of HBV replication and assessing responses to antiviral treatment. HBV DNA quantification measures virus replication and can be used as a prognosis indicator of liver disease and an index of response to antiviral drugs. The aim of this study was to compare the performances of three HBV DNA detection

Jean-Michel Pawlotsky; Anne Bastie; Isabelle Lonjon; Jocelyne Re´mire´; Françoise Darthuy; Claude-James Soussy; Daniel Dhumeaux

1997-01-01

352

A one step multiplex PCR assay for rapid screening of East Asian mtDNA haplogroups on forensic samples.  

PubMed

The mitochondrial DNA (mtDNA) haplogroup typing has become an essential tool to study human evolutionary history and to infer the matrilineal bio-geographic ancestry. In forensic field, the screening of mtDNA haplogroups by genotyping of mtDNA single nucleotide polymorphisms (SNPs) can help guarantee the quality of mtDNA sequence data as well as can reduce the need to sequence samples that do not match. Here, a multiplex mutagenically separated (MS) polymerase chain reaction (PCR) system was developed for simultaneous rapid detection of 14 coding region SNPs and one deletion motif representing common mtDNA haplogroups of East Asia. The multiplex MS PCR system we developed has the advantage of being a one step procedure that requires only a single PCR amplification with allele-specific primers and allowing straightforward designation of haplogroups along the branches of the phylogenetic tree. Therefore, it would be a simple, rapid, and reliable detection method useful for large-scale screening of mtDNA variations to determine East Asian mtDNA haplogroups. PMID:22981178

Lee, Hwan Young; Yoon, Jung Ah; Yang, Woo Ick; Shin, Kyoung-Jin

2012-09-13

353

Quantification of human mitochondrial DNA using synthesized DNA standards.  

PubMed

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

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

2011-09-02

354

Development of a quality, high throughput DNA analysis procedure for skeletal samples to assist with the identification of victims from the World Trade Center attacks.  

PubMed

The attacks on the World Trade Center (WTC) Towers on September 11, 2001, represented the single largest terrorist-related mass fatality incident in the history of the United States. More than 2,700 individuals of varied racial and ethnic background lost their lives that day. Through the efforts of thousands of citizens, including recovery workers, medical examiners, and forensic scientists, the identification of approximately 1,500 victims had been accomplished through June 2003 (the majority of these identifications were made within the first 8-12 months). The principal role of The Bode Technology Group (Bode) in this process was to develop a quality, high throughput DNA extraction and short tandem repeat (STR) analysis procedure for skeletal elements, and to provide STR profiles to the Office of the Chief Medical Examiner (OCME) in New York City to be used for identification of the victims. A high throughput process was developed to include electronic accessioning of samples, so that the numbering system of the OCME was maintained; rapid preparation and sampling of skeletal fragments to allow for the processing of more than 250 fragments per day; use of a 96-well format for sample extraction, DNA quantification, and STR analysis; and use of the Applied Biosystems 3100 and 3700 instrumentation to develop STR profiles. Given the highly degraded nature of the skeletal remains received by Bode, an advanced DNA extraction procedure was developed to increase the quantity of DNA recovery and reduce the co-purification of polymerase chain reaction (PCR) amplification inhibitors. In addition, two new STR multiplexes were developed specifically for this project, which reduced the amplicon size of the STR loci, and therefore, enhanced the ability to obtain results from the most challenged of samples. In all, the procedures developed allowed for the analysis of more than 1,000 skeletal samples each week. Approximately 13,000 skeletal fragments were analyzed at least once, for a total of more than 18,000 analyses, and greater than 8,000 of the skeletal samples produced STR results (65%). The percentage of successful results was low in relation to previous mass fatality incidents involving airline disasters. However, when this same process was applied to the analysis of skeletal remains from the American Airlines Flight 587 disaster that occurred on November 12, 2001, the success rate was in line with expected results (ie, greater than 92% of the skeletal remains produced results). This illustrated the quality aspects of the procedure and the degree of degradation that had occurred for the remains of the WTC victims. For future mass fatality incidents, the quality, high throughput procedures developed by Bode will allow for more rapid DNA analysis of victim remains, more rapid identification of victims, and thus more rapid return of remains to family members. PMID:12808717

Holland, Mitchell M; Cave, Christopher A; Holland, Charity A; Bille, Todd W

2003-06-01

355

Degradation of plasmid and plant DNA in water microcosms monitored by natural transformation and real-time polymerase chain reaction (PCR)  

Microsoft Academic Search

Extracellular DNA exists in the environment and can be taken up by competent bacterial cells, leading to horizontal gene transfer. The persistence of extracellular plasmid and plant DNA in water microcosms was monitored in this study. Water samples were two groundwater (GW1 and GW2) and one river water (RW) samples. Three treatments included: (1) intact, (2) 0.22?m filter-sterilized, and (3)

Bin Zhu

2006-01-01

356

Evaluation of DNA Extraction Techniques for Detecting Mycobacterium tuberculosis Complex Organisms in Asian Elephant Trunk Wash Samples?  

PubMed Central

Rapid and sensitive diagnostic assays for the detection of tuberculous mycobacteria in elephants are lacking. DNA extraction with PCR analysis is useful for tuberculosis screening in many species but has not been validated on elephant trunk wash samples. We estimated the analytical sensitivity and specificity of three DNA extraction methods to detect Mycobacterium tuberculosis complex organisms in trunk wash specimens. A ZR soil microbe DNA kit (ZR) and a traditional salt and ethanol precipitation (TSEP) approach were evaluated under three different treatment conditions: heat treatment, phenol treatment, and contamination with Mycobacterium avium. A third approach, using a column filtration method, was evaluated for samples contaminated with soil. Trunk wash samples from uninfected elephants were spiked with various concentrations of M. bovis cells and subjected to the described treatment conditions prior to DNA extraction. Extracted DNA was amplified using IS6110-targeted PCR analysis. The ZR and TSEP methods detected as low as 1 to 5 M. bovis cells and 10 M. bovis cells, respectively, per 1.5 ml of trunk wash under all three conditions. Depending on the amount of soil present, the column filtration method detected as low as 5 to 50 M. bovis cells per 1.5 ml of trunk wash. Analytical specificity was assessed by DNA extraction from species of nontuberculous mycobacteria and amplification using the same PCR technique. Only M. bovis DNA was amplified, indicating 100% analytical specificity of this PCR technique. Our results indicate that these DNA extraction techniques offer promise as useful tests for detection of M. tuberculosis complex organisms in elephant trunk wash specimens.

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

2011-01-01

357

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.

Iglesias-Guimarais, Victoria; Gil-Guinon, Estel; Gabernet, Gisela; Garcia-Belinchon, Merce; Sanchez-Osuna, Maria; Casanelles, Elisenda; Comella, Joan X.; Yuste, Victor J.

2012-01-01

358

Apoptotic DNA degradation into oligonucleosomal fragments, but not apoptotic nuclear morphology, relies on a cytosolic pool of DFF40/CAD endonuclease.  

PubMed

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

2012-01-17

359

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.

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

2013-01-01

360

Lack of reliability of nanotechnology in the of free plasma DNA in samples of patients with prostate cancer  

PubMed Central

Background Several studies seek biological markers that give diagnostic and degree of tumor development. The aim of this study was to validate the determination of plasma DNA using nanotechnology (Nanovue™-NV) in samples of 80 patients with prostate cancer. Methods Blood samples of 80 patients of the Urology Ambulatory of Faculdade de Medicina do ABC with prostate cancer confirmed by anatomical-pathology criteria were analyzed. DNA extraction was performed using a GFX TM kit (Amersham Pharmacia Biotech, Inc, USA) following the adapted protocol. Plasma was subjected to centrifugation. Results There was a big difference between the first and the second value obtained by NanoVue Only two samples had no differences between duplicates. Maximum difference between duplicates was 38??g/mL. Average variation between 51 samples was 10.29??g/mL, although 21 samples had differences above this average. No correlation was observed between pDNA obtained by traditional spectrophotometry and by nanotechnology. Conclusion Determination of plasma DNA by nanotechnology was not reproducible.

2013-01-01

361

Lack of reliability of nanotechnology in the of free plasma DNA in samples of patients with prostate cancer.  

PubMed

BACKGROUND: Several studies seek biological markers that give diagnostic and degree of tumor development. The aim of this study was to validate the determination of plasma DNA using nanotechnology (Nanovue™-NV) in samples of 80 patients with prostate cancer. METHODS: Blood samples of 80 patients of the Urology Ambulatory of Faculdade de Medicina do ABC with prostate cancer confirmed by anatomical-pathology criteria were analyzed. DNA extraction was performed using a GFX TM kit (Amersham Pharmacia Biotech, Inc, USA) following the adapted protocol. Plasma was subjected to centrifugation. RESULTS: There was a big difference between the first and the second value obtained by NanoVue Only two samples had no differences between duplicates. Maximum difference between duplicates was 38??g/mL. Average variation between 51 samples was 10.29??g/mL, although 21 samples had differences above this average. No correlation was observed between pDNA obtained by traditional spectrophotometry and by nanotechnology. CONCLUSION: Determination of plasma DNA by nanotechnology was not reproducible. PMID:23311763

Moreno, Ricardo; Delgado, Pamela O; Coelho, Patrícia G; Marsicano, Sarah R; Boas, Viviane Av; Azzalis, Ligia A; Junqueira, Virgínia Bc; Rocha, Katya C; de Abreu, Luiz Carlos; Valenti, Vitor E; Drezzet, Jefferson; Pereira, Edimar Cristiano; Fonseca, Fernando LA

2013-01-12

362

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

NASA Astrophysics Data System (ADS)

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

El-Metwaly, Nashwa M.; Refat, Moamen S.

2011-01-01

363

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

PubMed

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

El-Metwaly, Nashwa M; Refat, Moamen S

2010-09-18

364

Blood puncture as a nondestructive sampling tool to obtain DNA in frogs: comparison of protocols and survival analysis.  

PubMed

In molecular biology studies of Anura, nondestructive methods to obtain genetic material are needed as alternatives to toe clipping. This work evaluates a nondestructive method for sampling DNA from blood puncture, comparing the performance of three different extraction protocols (Qiagen Kit, Salting-out and Chelex). We collected 134 individuals of Eleutherodactylus johnstonei, extracting blood via puncture of the medial vein using commercial-grade glucometer lancets. We extracted 100-1880 ng DNA, finding no differences between the extraction protocols. We compared the quality of the resulting DNA through amplification and sequencing of the 16S mitochondrial gene. Amplification was successful for the three extraction protocols, although Chelex showed better performance, making it the most recommendable protocol for extraction of DNA from blood. The resulting sequences corresponded to those registered in the GenBank for this species. Additionally, we found no significant differences in survival or weight change between the individuals that were manipulated and a control group (mean survival 66.7% treated, 62.9% untreated). Data reveal that blood samples obtained by puncture are a convenient alternative to other tissues (phalange, buccal swab, liver) that have traditionally been used as DNA sources for anurans. The technique is applicable to small and large species, covering most anuran diversity, provides enough DNA for many genetic applications and produces no noticeable effect on the survival or performance, given that it does not affect the motor parts or the dexterity of the animals. PMID:22240248

Mendoza, A M; García-Ramírez, J C; Cárdenas-Henao, H

2012-01-12

365

Effect of enzyme additions on methane production and lignin degradation of landfilled sample of municipal solid waste.  

PubMed

Operation of waste cells as landfill bioreactors with leachate recirculation is known to accelerate waste degradation and landfill gas generation. However, waste degradation rates in landfill bioreactors decrease with time, with the accumulation of difficult to degrade materials, such as lignin-rich waste. Although, potential exists to modify the leachate quality to promote further degradation of such waste, very little information is available in literature. The objective of this study was to determine the viability of augmenting leachate with enzymes to increase the rate of degradation of lignin-rich waste materials. Among the enzymes evaluated MnP enzyme showed the best performance in terms of methane yield and substrate (lignin) utilization. Methane production of 200 mL CH(4)/g VS was observed for the MnP amended reactor as compared to 5.7 mL CH(4)/g VS for the control reactor. The lignin reduction in the MnP amended reactor and control reactor was 68.4% and 6.2%, respectively. PMID:21306893

Jayasinghe, P A; Hettiaratchi, J P A; Mehrotra, A K; Kumar, Sunil

2011-01-14

366

Smoking Related Carcinogen-DNA Adducts in Biopsy Samples of Human Urinary Bladder: Identification of N-(Deoxyguanosin-8-yl)-4Aminobiphenyl as a Major Adduct  

Microsoft Academic Search

The prevalence of covalent modifications to DNA (carcinogen-DNA adducts) in 42 human urinary bladder biopsy samples was investigated by 32P-postlabeling methods, with enhancement by both nuclease P1 treatment and 1-butanol extraction. Total mean carcinogen-DNA adduct levels and the mean levels of several specific adducts were significantly elevated in DNA samples of 13 current smokers, as opposed to 9 never smokers

Glenn Talaska; Amer Z. S. S. Al-Juburi; Fred F. Kadlubar

1991-01-01

367

Genomic DNA isolation of Acrocomia aculeata (Arecaceae) from leaf and stipe tissue samples for PCR analysis.  

PubMed

Macaw palm, Acrocomia aculeata is an oleaginous species of the Arecaceae family; it has been identified as one of the most promising plants for sustainable production of renewable energy, especially biodiesel. We developed an efficient protocol of genomic DNA extraction for A. aculeata using leaf and stipe tissues, based on the cationic hexadecyltrimethylammonium bromide method, and we evaluated the quantity, purity, and integrity of the resultant DNA. We also determined whether these procedures interfere with PCR amplification using SSR molecular markers. The lowest concentration of DNA was obtained from stipe tissues (135 ng/?L), while fresh leaf tissues provided the highest concentration of DNA (650 ng/?L). Good quality DNA was obtained from fresh leaf, lyophilized leaf, and stipe tissues (relative purity, 1.79-1.89 nm). Differences in quantity and quality of DNA extracted from different tissues did not interfere with general patterns of PCR amplification based on SSR markers. PMID:24085452

Lanes, E C M; Nick, C; Kuki, K N; Freitas, R D; Motoike, S Y

2013-09-23

368

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

2011-04-26

369

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

370

Human papillomavirus DNA detection and typing in male urine samples from a high-risk population from Argentina  

Microsoft Academic Search

The aim of the present study was to evaluate the first void urine (FVU) as a non-invasive sampling method for HPV detection and genotyping in a high-risk population. Men presenting with HPV associated penile lesions or HPV positive partners attending a urological department in La Plata, Argentina were enrolled for HPV detection and genotyping. DNA from 185 first-void urine samples

Carlos D. Golijow; Luis O. Pérez; Jennifer S. Smith; Martín C. Abba

2005-01-01

371

Collection of blood, saliva, and buccal cell samples in a pilot study on the Danish nurse cohort: comparison of the response rate and quality of genomic DNA.  

PubMed

In this study, we compared the response rates of blood, saliva, and buccal cell samples in a pilot study on the Danish nurse cohort and examined the quantity and quality of the purified genomic DNA. Our data show that only 31% of the requested participants delivered a blood sample, whereas 72%, 80%, and 76% delivered a saliva sample, buccal cell sample via mouth swabs, or buccal cell sample on FTA card, respectively. Analysis of purified genomic DNA by NanoDrop and agarose gel electrophoresis revealed that blood and saliva samples resulted in DNA with the best quality, whereas the DNA quality from buccal cells was low. Genotype and PCR analysis showed that DNA from 100% of the blood samples and 72% to 84% of the saliva samples could be genotyped or amplified, whereas none of the DNA from FTA cards and only 23% of the DNA from mouth swabs could be amplified and none of the DNA from swabs and 94% of the DNA from FTA cards could be genotyped. Our study shows that the response rate of self-collection saliva samples and buccal cell samples were much higher than the response rate of blood samples in our group of Danish nurses. However, only the quality of genomic DNA from saliva samples was comparable with blood samples as accessed by purity, genotyping, and PCR amplification. We conclude that the use of saliva samples is a good alternative to blood samples to obtain genomic DNA of high quality and it will increase the response rate considerably in epidemiologic studies. PMID:17932355

Hansen, Thomas V O; Simonsen, Mette K; Nielsen, Finn C; Hundrup, Yrsa Andersen

2007-10-01

372

Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding, genotyping, and disease diagnostics from fungal and oomycete samples.  

PubMed

The ubiquity, high diversity and often-cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one-step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single-copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe-based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol-chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol-chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use. PMID:23121735

Osmundson, Todd W; Eyre, Catherine A; Hayden, Katherine M; Dhillon, Jaskirn; Garbelotto, Matteo M

2012-11-03

373

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

374

HPV DNA Testing of the Residual Sample of Liquid-Based Pap Test: Utility as a Quality Assurance Monitor  

Microsoft Academic Search

HPV DNA testing of the residual sample volume of liquid-based Pap tests has been recommended as a way to determine the appropriate follow-up for women who have equivocal results in routine clinical screening. A major aspect of quality assurance in the cytopathology laboratory consists of correlation of smear interpretation with biopsy or conization results as mandated by CLIA '88. However,

Rosemary E. Zuna; William Moore; S. Terence Dunn

2001-01-01

375

Upscaled CTAB-Based DNA Extraction and Real-Time PCR Assays for Fusarium culmorum and F. graminearum DNA in Plant Material with Reduced Sampling Error  

PubMed Central

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

Brandfass, Christoph; Karlovsky, Petr

2008-01-01

376

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

2008-11-25

377

Anaerobic degradation of halogenated benzoic acids coupled to denitrification observed in a variety of sediment and soil samples  

Microsoft Academic Search

Denitrifying enrichment cultures utilizing monochlorinated benzoic acids as a carbon source were established using sediments and soils from a variety of sources as inocula. Enrichment cultures from most of the sites readily degraded 3- and 4chlorobenzoate within 2–4 weeks. Upon refeeding, 3- and 4-chlorobenzoate were rapidly depleted, and stable denitrifying cultures were obtained by repeated dilution and refeeding of the

Max M. Häggblom; Maria D. Rivera; Lily Y. Young

1996-01-01

378

A complete miniaturised genotyping system for the detection of single nucleotide polymorphisms in human DNA samples  

Microsoft Academic Search

We describe the design, construction and performance of a complete palm-sized device capable of rapid discrimination of a single nucleotide polymorphism in human DNA. The analysis of the DNA is achieved by means of an analysis of the temperature dependent change in fluorescence of hybridised fluorescent probes undergoing Förster resonance energy transfer. The developed device was used to accurately and

Mazher-Iqbal Mohammed; Graeme J. Sills; Martin J. Brodie; Elizabeth. M. Ellis; John. M. Girkin

2009-01-01

379

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

380

Quantification of the Flavonoid-Degrading Bacterium Eubacterium ramulus in Human Fecal Samples with a Species-Specific Oligonucleotide Hybridization Probe  

Microsoft Academic Search

To investigate the occurrence of the flavonoid-degrading bacterium Eubacterium ramulus in the human intestinal tract, an oligonucleotide probe designated S-S-E.ram-0997-a-A-18 was designed and validated, with over 90 bacterial strains representing the dominant described human fecal flora. Application of S-S-E.ram- 0997-a-A-18 to fecal samples from 20 subjects indicated the presence of E. ramulus in each individual tested in numbers from 4.4

RAINER SIMMERING; BRIGITTA KLEESSEN; MICHAEL BLAUT

1999-01-01

381

DNA microarray screening of differential gene expression in bone marrow samples from AML, non-AML patients and AML cell lines  

Microsoft Academic Search

This study used cDNA microarray technology to compare gene expression profiles in acute myeloblastic leukaemia (AML) with cDNA dot-blot and real time PCR analysis of cDNA transcripts to confirm array data. Patient AML marrow samples and AML cell lines were compared with normal\\/non-AML samples. Screening revealed five particular genes to be significantly differentially expressed across the sample groups. The migration-inhibitory

Emma Louise Court; M Ann Smith; Neil David Avent; John T Hancock; Lyn M Morgan; Atherton G Gray; J Graham Smith

2004-01-01

382

Lack of detection of human papillomavirus DNA in male urine samples.  

PubMed Central

OBJECTIVES--To evaluate polymerase chain reaction (PCR) methodology for the detection of urethral human papillomavirus (HPV) infection by examining urinary sediment from males. SETTING--Department of Genitourinary Medicine, Leeds General Infirmary. SUBJECTS--73 male patients attending for treatment of sexually transmitted diseases, including 14 patients with genital warts which did not involve the urethral meatus. METHODS--Urinary sediment was tested for HPV DNA and human beta globin gene DNA by PCR methodology. A consensus primer set capable of detecting a wide range of HPV types was used. PCR product was analysed by gel electrophoresis and ethidium bromide staining. RESULTS--HPV DNA was not detected in any of the specimens. Human beta globin gene DNA was identified in 40 of the 73 specimens (55%). CONCLUSIONS--Screening urinary sediment for HPV DNA by PCR methodology with analysis of PCR product by gel electrophoresis and ethidium bromide staining is probably unhelpful for studying the prevalence of urethral HPV infection in men.

Geddy, P M; Wells, M; Lacey, C J

1993-01-01

383

Development & evaluation of biotinylated DNA probe for clinical diagnosis of chikungunya infection in patients' acute phase serum & CSF samples.  

PubMed

Background & objectives: The resurgence of chikungunya virus (CHIKV) in the Indian Ocean Islands and India has drawn worldwide attention due to its explosive nature, high morbidity and complex clinico-pathological manifestations. The early confirmatory diagnosis of CHIKV is essential for management as well as control of unprecedented epidemics. The present study describes the development and evaluation of a highly sensitive and specific E1 structural gene specific biotinylated DNA probe for detection of chikungunya virus in clinical samples using a dot blot format. Methods: The complementary DNA (cDNA) of CHIKV was spotted on to nylon membrane. The membrane was subjected to prehybridization and hybridization and developed using a colour development solution containing DAB chromogen. Results: The CHIKV E1 specific DNA probe was highly sensitive detecting picogram levels of target nucleic acid. The comparative evaluation with SYBR Green I based real-time RT-PCR revealed 99 per cent accordance with a sensitivity and specificity of 99 and 98 per cent, respectively. The specificity of this assay was further confirmed through cross-reaction studies with confirmed dengue and Japanese encephalitis (JE) patient serum samples along with infected culture supernatant of Ross River and Saint Louis encephalitis and plasmid DNA of O'Nyong Nyong, Semlinki forest and Sindbis viruses. Interpretation & conclusion: The DNA probe reported in this study may be useful for specific, sensitive and confirmatory clinical diagnosis of chikungunya infection in acute phase human patient serum and CSF samples. This assay can also be used in the laboratory for quantification of viral antigen in cell culture supernatant for research purpose. PMID:24056565

Kumar, Jyoti S; Parida, Manmohan; Lakshmana Rao, P V

2013-07-01

384

Development & evaluation of biotinylated DNA probe for clinical diagnosis of chikungunya infection in patients' acute phase serum & CSF samples  

PubMed Central

Background & objectives: The resurgence of chikungunya virus (CHIKV) in the Indian Ocean Islands and India has drawn worldwide attention due to its explosive nature, high morbidity and complex clinico-pathological manifestations. The early confirmatory diagnosis of CHIKV is essential for management as well as control of unprecedented epidemics. The present study describes the development and evaluation of a highly sensitive and specific E1 structural gene specific biotinylated DNA probe for detection of chikungunya virus in clinical samples using a dot blot format. Methods: The complementary DNA (cDNA) of CHIKV was spotted on to nylon membrane. The membrane was subjected to prehybridization and hybridization and developed using a colour development solution containing DAB chromogen. Results: The CHIKV E1 specific DNA probe was highly sensitive detecting picogram levels of target nucleic acid. The comparative evaluation with SYBR Green I based real-time RT-PCR revealed 99 per cent accordance with a sensitivity and specificity of 99 and 98 per cent, respectively. The specificity of this assay was further confirmed through cross-reaction studies with confirmed dengue and Japanese encephalitis (JE) patient serum samples along with infected culture supernatant of Ross River and Saint Louis encephalitis and plasmid DNA of O’Nyong Nyong, Semlinki forest and Sindbis viruses. Interpretation & conclusion: The DNA probe reported in this study may be useful for specific, sensitive and confirmatory clinical diagnosis of chikungunya infection in acute phase human patient serum and CSF samples. This assay can also be used in the laboratory for quantification of viral antigen in cell culture supernatant for research purpose.

Kumar, Jyoti S.; Parida, Manmohan; Lakshmana Rao, P.V.

2013-01-01