Identification of a D8S1179 primer binding site mutation and the validation of a primer designed to recover null alleles

Abstract 

A population study of Chamorros and Filipinos using short tandem repeat (STR) loci amplified with the AmpFℓSTR^® Profiler Plus™ PCR amplification kit demonstrated an excess of observed homozygosity at the D8S1179 locus. Use of a different set of D8S1179 primers to type the same samples did not demonstrate an excess of homozygosity and showed discordant genotypes at the D8S1179 locus. A single point mutation, G-to-A transition, 16 nucleotides from the 3′ end of the reverse primer, was identified to cause allele dropout when using the AmpFℓSTR^® Profiler Plus™ primer set. An additional D8S1179 reverse primer specific for the variant was constructed resulting in the recovery of the null allele. The primer was included in the newly developed AmpFℓSTR^® Identifiler™ PCR amplification kit. No deleterious effects or non-specific peaks were observed in validation experiments evaluating primer concentration, Mg²⁺ concentration, annealing temperature and population samples.

Keywords:  D8S1179, Primer binding site mutation, AmpFℓSTR^®, Multiplex, PCR, STR, Validation