Detection of a primer-binding site polymorphism for the STR locus D16S539 using the Powerplex 1.1 system and validation of a degenerate primer to correct for the polymorphism

Quality assurance samples submitted from the NCSBI as part of a contract with TBTG to outsource DNA Database samples showed unexpected discrepancies for the locus D16S539 when all other loci yielded identical results. Discrepancies observed included allele drop out and an imbalance in sister alleles...

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Bibliographic Details
Published in:	Journal of forensic sciences Vol. 47; no. 2; pp. 345 - 349
Main Authors:	Nelson, Mark S, Levedakou, Eleni N, Matthews, Janis R, Early, Buddy E, Freeman, David A, Kuhn, Cynthia A, Sprecher, Cynthia J, Amin, Ashima S, McElfresh, Kevin C, Schumm, James W
Format:	Journal Article
Language:	English
Published:	United States Wiley Subscription Services, Inc 01-03-2002
Subjects:	Allelic Imbalance - genetics Binding Sites Black or African American Black People - genetics Blood Stains Chromosomes, Human, Pair 16 - genetics Deoxyribonucleic acid DNA DNA Fingerprinting - methods DNA Fingerprinting - standards DNA Primers - analysis Forensic sciences Gene Frequency Genotype Homozygote Humans Mutation Nucleic Acid Amplification Techniques - methods Point Mutation Polymorphism, Genetic Reproducibility of Results Tandem Repeat Sequences - genetics White People - genetics
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Summary:	Quality assurance samples submitted from the NCSBI as part of a contract with TBTG to outsource DNA Database samples showed unexpected discrepancies for the locus D16S539 when all other loci yielded identical results. Discrepancies observed included allele drop out and an imbalance in sister alleles with samples returned from TBTG. This led to a comprehensive review of the technical procedures used between the two laboratories to determine the cause of the discrepancies noted for the locus D16S539, since both laboratories were using the PowerPlex 1.1 typing kit from the Promega Corporation. The NCSBI and the TBTG utilize different extraction methods (organic extraction vs. FTA) and amplification conditions (AmpliTaq vs AmpliTaq Gold), respectively, so the exact cause of discrepancy observed was not immediately apparent. Experiments at the NCSBI associated the observed allele drop out and the imbalance of the sister alleles with the use of AmpliTaq Gold and a hot start procedure. Sequencing data revealed that a point mutation resides on the D16S539 primer-binding site that reaches polymorphic levels in African-American populations. This led to the development of a degenerate primer by the Promega Corporation to detect "missing" alleles when AmpliTaq Gold is used. The degenerate primer was then thoroughly tested to show its efficacy in detecting the "true" D16S539 profile when used.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 ObjectType-Undefined-3
ISSN:	0022-1198 1556-4029
DOI:	10.1520/jfs15255j