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Biomedical subjects

Robert W Allen

Publications and source records attributed to Robert W Allen.

5 recordsLinked to original sources
PubMed2006

Quantitation of human genomic DNA through amplification of the amelogenin locus.

An alternate method for quantitation of human genomic DNA is presented. Quantitative template amplification technology (abbreviated "Q-TAT") estimates the quantity of human DNA present in an extract by comparing fluorescence in X and Y amplicons produced from unknowns with fluorescence in a standard curve amplified from known quantities of reference DNA. Q-TAT utilizes PCR and electrophoresis with fluorescent detection/quantitation, precluding the need for new instrumentation, methodology, or quality assurance associated with slot-blot or real-time PCR. In a comparison study incorporating shared samples, Q-TAT was found to be more sensitive than widely used slot-blot methods but somewhat less sensitive than real-time PCR. Among samples containing DNA concentrations ranging from 100 pg/microL to 2-4 ng/microL, Q-TAT produced DNA concentration estimates that agreed reasonably well with either Quantiblot or real-time PCR. Q-TAT was reproducible with a typical coincidence of variation of about 35%. Quantitation of human DNA in this study involved summing fluorescence in X and Y amplicons in unknowns and quantitation standards. However, analyzing fluorescence in X and Y amplicons individually could allow estimates of male and female DNA present in mixtures to be made. Moreover, since X and Y amplicons exhibit sizes of 210 and 216 bp, respectively, the integrity as well as the concentration of the genomic DNA template can be assessed. Q-TAT represents an alternate method useful for the quantitation of human genomic DNA prior to amplification of STR loci used for identity testing purposes. The method uses existing equipment and procedures in conjunction with a well-characterized DNA standard to produce concentration estimates for unknowns that reliably produce STR profiles suitable for analysis.

Amelogenin↗
PubMed2004

Characteristics of mutations at the D5S818 locus studied with a tightly linked marker.

BACKGROUND: Mutations to STR alleles are not well understood in terms of the mechanism(s) underlying such mutations or their relative frequency among different alleles at the locus. STUDY DESIGN AND METHODS: A cytosine/thymosine (C/T) polymorphism was discovered 13 nucleotides upstream from the 5'-end of the tandem array of the D5S818 locus (-13SNP). The -13SNP coincidentally creates a restriction site polymorphism for the restriction endonuclease SnaBI that is tightly linked to the tandem array of D5S818. Forty D5S818 addition/deletion mutations in the tandem array were characterized with RFLP analysis with SnaBI. RESULTS: Among 40 mutations studied, 34 (approximately 85%) were of paternal origin, 1 ( approximately 3%) was of maternal origin, and 5 (approximately 13%) had an unclear lineage of origin. In 26 cases where the magnitude of the repeat change could be determined, 23 (88%) involved changes of a single repeat unit, whereas 3 (12%) involved changes of 2 or more repeats. The number of additions to the tandem array was roughly equal to the number of deletions from the tandem array. In 19 instances it was possible to identify the parental allele that underwent the mutation. Alleles 13 and 14 were prone to mutation, whereas Allele 11 was resistant. Thus, there is an unequal sensitivity to mutation among D5S818 alleles. CONCLUSIONS: The use of the linked -13SNP marker has revealed several features of mutations at the D5S818 locus: 1) Single repeat changes are the most commonly observed. 2) Mutations are more likely in the paternal lineage. 3) Not all alleles undergo mutation with equal frequency.

Adult↗
PubMed2004

Resolution of a serum sample mix-up through the use of short tandem repeat DNA typing.

BACKGROUND: A sample mix-up occurred in a tissue procurement laboratory in which aliquots of serum from two tissue donors were accidentally mislabeled. The clues to the apparent mixup involved discrepant Hepatitis C test results. In an attempt to resolve the apparent mix up, DNA typing was performed using serum samples as a possible source of genomic DNA. STUDY DESIGN AND METHODS: Two hundred microliter aliquots of two reference sera and aliquots prepared from them were subjected to DNA extraction. PCR amplification of 9 STR loci was performed on the extracts and amplicons were analyzed by capillary electrophoresis. RESULTS: About 1 microg/ml of DNA was recovered from all serum samples and was of sufficient quality to direct the amplification of most, if not all STR loci allowing the mislabeled specimens to be traced to the proper tissue donor. CONCLUSIONS: Serum is a useful source of genomic DNA for STR analysis in situations in which such samples are the only source of DNA for testing. Interestingly, one of the tissue donors on life support and repeatedly receiving blood products, exhibited a mixed DNA profile indicative of the presence of DNA from multiple individuals in the bloodstream.

DNA↗
PubMed2002

Paternity Testing Commission of the International Society of Forensic Genetics: recommendations on genetic investigations in paternity cases.

The International Society for Forensic Genetics (ISFG) has established a Paternity Testing Commission (PTC) with the purpose of formulating international recommendations concerning genetic investigations in paternity testing. The PTC recommends that paternity testing be performed in accordance with the ISO 17025 standards. The ISO 17025 standards are general standards for testing laboratories and the PTC offers explanations and recommendations concerning selected areas of special importance to paternity testing.

Equipment and Supplies↗