Evaluation of a method for typing the microsatellite D12S391 locus using a new primer pair and capillary electrophoresis.

We describe a modified method for typing a polymorphic microsatellite D12S391 locus by PCR using a newly designed primer pair. This primer pair produces shorter D12S391 amplified fragments (104-156 bp) than the primer pair originally described by Lareu et al. (209-261 bp). The detection system for the D12S391 locus using the new primer pair and capillary electrophoresis (CE) analysis was evaluated using various forensic samples. The typing results from 70 DNA samples using the new primer pair and the original primer pair were completely identical. One hundred twenty-five amplified fragments from D12S391 alleles were sized correctly within +/- 0.25 bp of the D12S391 allelic ladder. A rare allele, 19.3, previously found only in Caucasians, was found for the first time in a Japanese subject, and it was clearly distinguished from allele 20 by the CE analysis. This detection system was sensitive and could detect D12S391 types from 16 pg of genomic DNA, and from a minor component at a ratio of 1:10 in mixed samples. This system was more useful for the analysis of degraded DNA than was the method using the original primer pair, and could detect D12S391 types from bloodstains that had been stored for 26 years. In addition, the specificity of the method was demonstrated using nonhuman DNA.