Single-tube amplification of multiple loci for STR DNA typing is a common practice in forensic laboratories. Commercial suppliers are now releasing "megaplex" STR kits, allowing for the co-amplification of up to ten loci. It is commonly perceived by PCR product developers that the level of difficulty in achieving a good balance for color, locus and allelic balance is increasing with the number of loci being co-amplified. Therefore, we have set out to assess the characteristics of the pre-release Profiler-II kit (D3S1358, vWA, FGA; Amelogenin, D8S1179, D21S11, D8S51; D13S317, D7S820) under various templates ratios and modification of PCR cycling conditions. We have carried out dilutions and profile mixture series, at 3 different final PCR reaction volumes. In our laboratory, a two- and three-fold decrease in reaction volume coupled with a 90-second annealing segment did translate into a two- and three-fold increase respectively in yield, and allowed detection down to 0.1-0.2 ng of template DNA. Two-profile mixtures at reduced reaction volume allowed for detection of 10% minor profiles in a sample containing 1 ng of template DNA; whereas a 30% minor component was not routinely detected in a full reaction volume (50ml). Three-profile mixtures at reduced reaction volumes showed the benefits afforded by higher yields and the use of more input template DNA, but the measured ratio of non-shared peaks did stray away from expected values.

Although the 15ml reaction volume is affording the best overall detection capability, we have opted for a more conservative 25ml reaction volume format to lessen the impact of reduced reactant pool on template-independent extra-A addition, as well as maintaining a reasonable dilution factor for potential inhibitors of the amplification reaction.