Newly designed multiplex amplification and genotyping system at four pentanucleotide repeat STR loci useful for degraded mixed DNA specimens

Abstract We constructed a multiplex PCR and genotyping system selecting four pentanucleotide STR loci (Penta E, Penta D, Penta B and D10S2325) with multi-colored fluorescently labeled primer sets that are newly designed in order to genotype from mixed specimens, especially for degraded DNA samples, of which the amplicon sizes are smaller than about 200 bp. The allele frequencies at these four STR loci were calculated in a Japanese population. Sequence analysis was also performed for all alleles at these all STR loci observed in the Japanese population, and the allele distributions at those four loci did not deviated from HWE. The heterozygosities and the power of discriminations (PD) at all the four loci were more than 0.80 and 0.93, respectively. Furthermore, the present study provides a statistical standard to decide whether a one-repeat small peak from a main peak is the stutter peak or the peak originated from mixed individual sample.