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P S Walsh

Publications and source records attributed to P S Walsh.

17 recordsLinked to original sources
PubMed2002

TWGDAM validation of AmpFlSTR PCR amplification kits for forensic DNA casework.

Laboratory procedures used in short tandem repeat (STR) analysis were subjected to various scenarios that assessed reliability and identified potential limitations. These validation studies were designed as recommended by the Technical Working Group on DNA Analysis Methods (TWGDAM) and the DNA Advisory Board (DAB) (17,18). Various DNA samples were amplified by the polymerase chain reaction (PCR) using AmpFlSTR PCR Amplification Kits (i.e., AmpFlSTR Green I, Profiler, Profiler Plus, and COfiler kits), detected with ABI Prism instrumentation, and analyzed using GeneScan and Genotyper software. Data acquired in these studies reinforced an existing body of knowledge and expertise regarding application and interpretation of STR typing in the forensic science community. Consistent STR genotypes were detected in various body tissues and fluids. Inter-laboratory comparisons produced concordant genotype results. Quantitative interpretational aids for DNA mixtures were characterized. Ability of the typing systems to type potentially compromised samples reliably was evaluated. Nonprobative case evidentiary DNA was successfully amplified, genotyped, and interpreted. Potential limitations or cautionary factors in the interpretation of minimal fluorescence intensity were demonstrated. Differential amplification between loci was observed when PCR was inhibited; preferential amplification typically was not. Single AmpFlSTR locus amplification did not offer consistent benefit over AmpFlSTR multiplexing, even in cases of DNA degradation or PCR inhibition. During rigorous evaluation, AmpFlSTR PCR Amplification Kits reproducibly yielded sensitive and locus-specific results, as required in routine forensic analyses.

DNA↗
PubMed2001

Sequence variation in humans and other primates at six short tandem repeat loci used in forensic identity testing.

A large number of alleles from the six different short tandem repeat (STR) loci FGA, D3S1358, vWA, CSF1PO, TPOX and TH01, used in human identity testing were sequenced to provide support for the robustness of fluorescent STR DNA typing by allele size. Sequence information for some of these loci (FGA, vWA, TH01) is an extension of published work, whereas no extensive sequence information is available with respect to the D3S1358, CSF1PO, and TPOX loci. Sequencing of alleles at each locus has provided quantitative data with respect to the true nucleotide length of common alleles, and of alleles that vary in length from the common alleles. All alleles that were identified as "off-ladder" alleles through fluorescent typing at these STR loci have proven to be true length variant alleles. Sequencing at the D3S1358 and CSF1PO loci allowed for the establishment of a common nomenclature for these loci. A correlation between percent stutter and the length of the core tandem repeat is demonstrated at the FGA locus. Alleles in which the core tandem repeat is interrupted by a repeat unit of different sequence have a reduced percent stutter. DNA samples from three non-human primates (chimpanzee, orangutan, and gorilla) were compared to the human sequences, and shown to differ markedly across loci with respect to their homology. The effects of primer binding site mutations on the amplification efficiency at a particular locus, and methods used to interpret amplification imbalance of heterozygous alleles at a locus is also addressed.

Animals↗
PubMed2001

Regional Italian allele frequencies at nine short tandem repeat loci.

An Italian population study was performed on the loci D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S820 and a portion of the X-Y homologous gene Amelogenin for gender determination using the AmpFlSTR Profiler kit (PE Biosystems, Foster city, CA). This study was done on a population of 618 unrelated Italian individuals from 18 regions in Italy (except for Valle d'Aosta and Sardinia) to determine allele frequencies for each STR locus, and to evaluate STR technology for developing an Italian Offender DNA database.

Alleles↗
PubMed2000

Practical applications of genotypic surveys for forensic STR testing.

Legitimate genotype frequency estimation for multiallelic loci relies on component allele frequencies, as population surveys represent only a fraction of possible DNA profiles. Multilocus genotypes from two ethnic human populations, African American (n=195) and U.S. Caucasian (n=200), were compiled at 13 STR loci that are used worldwide in forensic investigation (D3S1358, vWA, FGA, D16S539, TH01, TPOX, CSF1PO, D8S1179, D21S11, D18S51, D5S818, D13S317, and D7S820). Sex-specific AmpFlSTR multiplexes provided stringent PCR-based STR typing specifically optimized for multicolor fluorescence detection. Heterozygosity at each STR locus ranged from 0.57 to 0.89 and encompassed from seven (TH01) to twenty-one (D21S11) alleles. Homozygosity tests, tests based on the distinct numbers of observed homozygous and heterozygous classes, log likelihood ratio tests, and exact tests assessed that the degree of divergence from theoretical Hardy-Weinberg proportions for all 13 STRs does not have practical consequence in genotype frequency estimation. Departures from linkage equilibrium, between loci, that imposed significance to forensic calculations were not indicated by observed variance of the number of heterozygous loci or Karlin interclass correlation tests. For forensic casework, reliable multilocus profile estimates may be obtained from the product of component genotype frequencies, each calculated through application of the Hardy-Weinberg equation to population database allele frequency estimates reported here. The average probability that two randomly selected, unrelated individuals possess an identical thirteen-locus DNA profile was one in 1.8x10(15) African Americans and one in 3.8x10(14) U.S. Caucasians.

Black or African American↗
PubMed1998

Genotyping of forensic short tandem repeat (STR) systems based on sizing precision in a capillary electrophoresis instrument.

Automated fluorescence analysis of polymerase chain reaction (PCR)-amplified short tandem repeat (STR) systems by capillary electrophoresis (CE) is becoming an established tool both in forensic casework and in the implementation of both state and national convicted offender DNA databases. A new capillary electrophoresis instrument, the ABI Prism 310 Genetic Analyzer, along with the Performance Optimized Polymer 4 (POP-4) provides an automated and precise method for simultaneously analyzing ten fluorescently labeled STR loci from a single PCR amplification kit, which provides a power of discrimination of approximately one in five billion from a single PCR amplification. Data are presented on sizing precision, sizing accuracy, and resolution for the STR loci in the AmpFlSTR Profiler kit. Sizing accuracy is highly dependent on the electrophoresis system, and therefore the reporting of alleles based on the nucleotide size obtained from an electrophoresis system is not recommended for forensic work. The precision of the 310 capillary electrophoresis system, coupled with software developed for automated genotyping of alleles based on the use of an allelic ladder, allows for accurate genotyping of STR loci. Sizing precision of < or = 0.16 nucleotide standard deviation was obtained with this system, thus allowing for accurate genotyping of length variants that differ in length by a single nucleotide.

Alleles↗
PubMed1998

TWGDAM validation of the AmpFISTR blue PCR amplification kit for forensic casework analysis.

Studies were performed as recommended by the Technical Working Group on DNA Analysis Methods (TWGDAM) committee to validate the AmpFISTR Blue PCR Amplification Kit for forensic casework applications. The kit coamplifies the tetranucleotide short tandem repeat (STR) loci D3S1358, vWA, and FGA. The dye-labeled amplification products were electrophoresed and detected directly using the ABI PRISM 377 DNA Sequencer or the 310 Genetic Analyzer. CEPH family studies demonstrated Mendelian inheritance of these loci and probability of identity values from population studies were 1/4,830 (African-American), 1/5,479 (U.S. Caucasian), and 1/3,443 (U.S. West Coast Hispanic). In all studies examining different body tissues and fluids, the expected genotypes were observed. Studies to determine and test the PCR reagent components and thermal cycling parameters demonstrated specificity, sensitivity, and balance over a wide range of conditions. Reliable results were obtained from DNA quantities as low as 0.25 ng. A variety of environmental studies were performed, as forensic samples are often exposed to different environmental conditions and substances which may degrade DNA or inhibit the amplification process. Highly degraded samples demonstrated that FGA was the first locus to become undetectable, followed by vWA, and then D3S1358; this is the expected pattern according to locus size. In studies of PCR inhibition, the pattern in which the loci became undetectable was different; FGA was the first locus to become undetectable, followed by D3S1358, and then vWA. Single versus multiple locus amplifications revealed no benefit to single locus analysis, even in cases of degradation or inhibition. The occurrence of preferential amplification was very rare, particularly in noncompromised, unmixed samples. Artifact peaks were not observed in any instance. Mixture studies confirmed the ability to detect mixed DNA samples and included the characterization of stutter and peak height ratios; the limit of detection was 1:10 for 1 ng total genomic DNA and 1:30 for 5 ng. DNA extracted from nonprobative case evidence was successfully amplified and genotyped. All such studies indicate that the AmpFISTR Blue PCR Amplification Kit will reproducibly yield specific and sensitive results.

Animals↗
PubMed1996

Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA.

The PCR amplification of tetranucleotide short tandem repeat (STR) loci typically produces a minor product band 4 bp shorter than the corresponding main allele band; this is referred to as the stutter band. Sequence analysis of the main and stutter bands for two sample alleles of the STR locus vWA reveals that the stutter band lacks one repeat unit relative to the main allele. Sequencing results also indicate that the number and location of the different 4 bp repeat units vary between samples containing a typical verses low proportion of stutter product. The results also suggest that the proportion of stutter product relative to the main allele increases as the number of uninterrupted core repeat units increases. The sequence analysis and results obtained using various DNA polymerases appear to support the slipped strand displacement model as a potential explanation for how these stutter products are generated.

Alleles↗
PubMed1992

A rapid chemiluminescent method for quantitation of human DNA.

A sensitive and simple method for the quantitation of human DNA is described. This method is based on probe hybridization to a human alpha satellite locus, D17Z1. The biotinylated probe is hybridized to sample DNA immobilized on nylon membrane. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for chemiluminescent detection using a luminol-based reagent and X-ray film. Less than 150 pg of human DNA can easily be detected with a 15 minute exposure. The entire procedure can be performed in 1.5 hours. Microgram quantities of nonhuman DNA have been tested and the results indicate very high specificity for human DNA. The data on film can be scanned into a computer and a commercially available program can be used to create a standard curve where DNA quantity is plotted against the mean density of each slot blot signal. The methods described can also be applied to the very sensitive determination of quantity and quality (size) of DNA on Southern blots. The high sensitivity of this quantitation method requires the consumption of only a fraction of sample for analysis. Determination of DNA quantity is necessary for RFLP and many PCR-based tests where optimal results are obtained only with a relatively narrow range of DNA quantities. The specificity of this quantitation method for human DNA will be useful for the analysis of samples that may also contain bacterial or other non-human DNA, for example forensic evidence samples, ancient DNA samples, or clinical samples.

Animals↗
PubMed1992

Simultaneous amplification and detection of specific DNA sequences.

We have enhanced the polymerase chain reaction (PCR) such that specific DNA sequences can be detected without opening the reaction tube. This enhancement requires the addition of ethidium bromide (EtBr) to a PCR. Since the fluorescence of EtBr increases in the presence of double-stranded (ds) DNA an increase in fluorescence in such a PCR indicates a positive amplification, which can be easily monitored externally. In fact, amplification can be continuously monitored in order to follow its progress. The ability to simultaneously amplify specific DNA sequences and detect the product of the amplification both simplifies and improves PCR and may facilitate its automation and more widespread use in the clinic or in other situations requiring high sample throughput.

Alleles↗
PubMed1992

Preferential PCR amplification of alleles: mechanisms and solutions.

The preferential PCR amplification of one allele relative to another in a heterozygous sample could result in an incorrect or ambiguous genetic typing of that sample. There are several mechanisms that could potentially lead to such preferential PCR amplification. First, preferential amplification can result from significant GC% differences between alleles if the conditions of the reaction (denaturation temperature (Tden), duration at the Tden' salt and co-solvent concentrations, etc.) allow the denaturation of one allele but not the other (differential denaturation). For example, the DQa1.1, -1.2, and -1.3 alleles of the HLA-DQa locus do not amplify at a Tden < 89 degrees C; these same conditions still allow amplification of the DQa2, -3, and -4 alleles. However, no differences in amplification efficiency were found between the different HLA-DQa alleles when the Tden was set at the recommended Tden of 94 degrees C, even after as many as 102 cycles of amplification. Second, for PCR-based genetic typing systems in which the PCR products from different alleles differ in length, preferential amplification of the shorter allelic product can occur. Experiments in which the variable number tandem repeat (VNTR) marker D17S5 (YNZ22) was amplified under various conditions suggest that the smaller allelic products are amplified preferentially when Taq polymerase is limiting. Preferential amplification of VNTR alleles can also occur if the target DNA is sufficiently degraded. Third, when the initial number of genomes sampled is very small, stochastic fluctuation in the number of copies of each allele can result in what appears to be preferential amplification. Finally, less efficient priming of DNA synthesis of one allele versus another can occur because of mismatches between the primer and the specific allelic template, resulting in preferential amplification of the other allele. General strategies to avoid preferential amplification are discussed.

Alleles↗
PubMed1992

Polymerase chain reaction (PCR) amplification and human leukocyte antigen (HLA)-DQ alpha oligonucleotide typing on biological evidence samples: casework experience.

The polymerase chain reaction (PCR) method of specific gene amplification was used in casework to synthesize millions of copies of the polymorphic second exon of the human leukocyte antigen (HLA)-DQ alpha (or DQA1) locus from a variety of evidence samples. The HLA-DQ alpha allelic variants in the amplified deoxyribonucleic acid (DNA) were determined in a rapid non-radioactive test by hybridization to sequence-specific oligonucleotide probes in both the dot-blot and reverse dot-blot formats. This genetic typing system has been subjected to blind proficiency testing; the performance of this test in the analysis of experimentally mixed samples was also evaluated. As of August 1990, over 250 cases have been tested and more than 2000 individual evidence (bloodstains, semen stains, individual hairs, bone fragments, and tissue sections) and reference samples have been analyzed. The first 198 of these cases are summarized in this paper; in 65% of the cases with conclusive results a suspect was included, and in 35%, all suspects were excluded. Individual cases as well as some of the general issues relating to forensic science analysis and this genetic typing system are discussed. The high rate of exclusion reported here combined with the ability of PCR to type old evidence samples suggests the relevance of this genetic test for postconviction review; two cases in which the convicted suspect was excluded are discussed.

Adolescent↗
PubMed1991

Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material.

Procedures utilizing Chelex 100 chelating resin have been developed for extracting DNA from forensic-type samples for use with the PCR. The procedures are simple, rapid, involve no organic solvents and do not require multiple tube transfers for most types of samples. The extraction of DNA from semen and very small bloodstains using Chelex 100 is as efficient or more efficient than using proteinase K and phenol-chloroform extraction. DNA extracted from bloodstains seems less prone to contain PCR inhibitors when prepared by this method. The Chelex method has been used with amplification and typing at the HLA DQ alpha locus to obtain the DQ alpha genotypes of many different types of samples, including whole blood, bloodstains, seminal stains, buccal swabs, hair and post-coital samples. The results of a concordance study are presented in which the DQ alpha genotypes of 84 samples prepared using Chelex or using conventional phenol-chloroform extraction are compared. The genotypes obtained using the two different extraction methods were identical for all samples tested.

Chelating Agents↗
PubMed1991

Report of the blind trial of the Cetus Amplitype HLA DQ alpha forensic deoxyribonucleic acid (DNA) amplification and typing kit.

The AmpliType HLA DQ alpha forensic DNA amplification and typing kit is designed for the qualitative analysis of the human leukocyte antigen (HLA) DQ alpha alleles present in deoxyribonucleic acid (DNA) extracted from forensic samples. The AmpliType kit is the first forensic DNA typing product based on the GeneAmp polymerase chain reaction (PCR) process. The kit was evaluated by five forensic science laboratories (test sites) to assess their ability to perform DNA typing using PCR on sample types typically encountered by forensic laboratories. None of the DNA-containing samples was mistyped. Of the 180 DNA-containing samples analyzed, results were reported for 178 (98.9%). Of the 178 samples with results, all were correctly typed. Two sites did not report a result for one sample each. Four of the five laboratories experienced no significant levels of contamination in the DNA-containing samples. At the one site with the highest number of DNA-containing samples with contamination, the typing results were not compromised. This site was able to correct the contamination problem through simple procedural changes and stricter attention to sterile technique. Blank controls were important to monitor contamination. In conclusion, the trial demonstrated that forensic science laboratories are capable of setting up a PCR-based DNA typing laboratory and successfully using the AmpliType HLA DQ alpha forensic DNA amplification and typing kit to analyze forensic samples.

Alleles↗
PubMed1989

Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes.

The analysis of DNA for the presence of particular mutations or polymorphisms can be readily accomplished by differential hybridization with sequence-specific oligonucleotide probes. The in vitro DNA amplification technique, the polymerase chain reaction (PCR), has facilitated the use of these probes by greatly increasing the number of copies of target DNA in the sample prior to hybridization. In a conventional assay with immobilized PCR product and labeled oligonucleotide probes, each probe requires a separate hybridization. Here we describe a method by which one can simultaneously screen a sample for all known allelic variants at an amplified locus. In this format, the oligonucleotides are given homopolymer tails with terminal deoxyribonucleotidyltransferase, spotted onto a nylon membrane, and covalently bound by UV irradiation. Due to their long length, the tails are preferentially bound to the nylon, leaving the oligonucleotide probe free to hybridize. The target segment of the DNA sample to be tested is PCR-amplified with biotinylated primers and then hybridized to the membrane containing the immobilized oligonucleotides under stringent conditions. Hybridization is detected nonradioactively by binding of streptavidin-horseradish peroxidase to the biotinylated DNA, followed by a simple colorimetric reaction. This technique has been applied to HLA-DQA genotyping (six types) and to the detection of Mediterranean beta-thalassemia mutations (nine alleles).

Alleles↗
PubMedDate not supplied

HLA-DR, DQ and DP typing using PCR amplification and immobilized probes.

A simple, rapid, and precise method of typing HLA class II polymorphism would be valuable in the areas of disease susceptibility, tissue transplantation, individual identification and anthropological genetics. Here we describe a method of analysing class II sequence polymorphism based on polymerase chain reaction (PCR) amplification and hybridization with oligonucleotide probes. One valuable property of sequence-based HLA typing strategies, like oligonucleotide probe hybridization, is that they reveal how and where two alleles differ, not simply that they can be operationally distinguished. The nature and location of HLA polymorphisms appears to be critical in disease association studies and are likely to be important in tissue typing for transplantation. New alleles at the DRB1, DPB1 and DQB1 loci are likely to be identified as this technology is applied to more and more samples, particularly in non-Caucasian ethnic groups. A new allele is uncovered as an unusual pattern of probe binding and then confirmed by sequencing. This pattern is observed because class II polymorphism is localized to specific regions and virtually all 'new' alleles have polymorphisms in the region of probe binding. Obviously, any new allele with a new polymorphic sequence in a region for which typing probes are not available would not be revealed by oligonucleotide typing. With the PCR primers and probes described here, 7 DQA1 alleles, 15 DQB1 alleles, 18 DPB1 alleles, and 32 DRB1 alleles are distinguished. Additional primers and/or probes can, of course, increase the allelic discrimination of oligonucleotide dot blot typing. These horseradish peroxidase (HRP)-labelled oligonucleotide probes are stable (greater than 2 years when stored at 4 degrees C) and the typing system is simple and robust. Over 500 samples from the CEPH pedigrees (unpublished data; A. B. Begovich, et al., manuscript in preparation) and greater than 1000 unrelated samples have been typed by this procedure. Although this dot blot/oligonucleotide hybridization procedure is a powerful and precise method of HLA class II typing, the complexity of the procedure increases as the number of probes required for analysis increases. The reverse dot blot method, based on an array of immobilized probes, allows the typing of individual samples in one single hybridization reaction. In this approach, a panel of unlabelled oligonucleotides are immobilized to a nylon membrane. The PCR product is labelled during the amplification reaction by using biotinylated primers and hybridized to the membrane. The presence of bound PCR product specifically hybridized to a given probe is detected using streptavidin-HRP conjugates and either chromogenic or chemiluminescent substrates.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗