Validation of a male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex $

Abstract Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex ® Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex ® Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24–28 cycles) could be modified to match different substrates (such as direct amplification of FTA ® paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 μL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5–2× primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1 mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2–4 °C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 °C and significant locus dropout with a 4 °C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with ≥125 pg of male template with partial and/or complete profiles observed using 30–62.5 pg of DNA; (k) analysis of ≤500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with ≤1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10–20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex ® Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.