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 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.
Nelson
Mark S
MS
North Carolina State Bureau of Investigation, Molecular Genetics Section, Raleigh 27603, USA. Mnelson@mail.jus.state.nc.us
Levedakou
Eleni N
EN
Matthews
Janis R
JR
Early
Buddy E
BE
Freeman
David A
DA
Kuhn
Cynthia A
CA
Sprecher
Cynthia J
CJ
Amin
Ashima S
AS
McElfresh
Kevin C
KC
Schumm
James W
JW
D022981:Allelic Imbalance / Q000235:genetics; D001665:Binding Sites; D044383:Black People / Q000235:genetics; D001801:Blood Stains; D002885:Chromosomes, Human, Pair 16 / Q000235:genetics; D016172:DNA Fingerprinting / Q000379:methods* / Q000592:standards; D017931:DNA Primers / Q000032:analysis*; D005787:Gene Frequency; D005838:Genotype; D006720:Homozygote; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D017354:Point Mutation; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D020080:Tandem Repeat Sequences / Q000235:genetics*; D044465:White People / Q000235:genetics; D001741:Black or African American
D017931:DNA Primers