Demonstration of Rapid Multiplex PCR Amplification Involving 16 Genetic Loci
Peter M. Vallone, Carolyn R. Steffen, John M. Butler
Current forensic DNA typing is conducted in approximately eight to ten hours with steps including DNA extraction, quantitation, polymerase chain reaction (PCR) amplification of multiple short tandem repeat (STR) loci, capillary electrophoresis separation with fluorescence detection and data analysis, and DNA profile interpretation. The PCR amplification portion of the workflow typically takes approximately three hours with standard thermal cycling protocols. Here we demonstrate a rapid cycling protocol that amplifies 15 STR loci and the sex-typing marker amelogenin from either Identifiler or PowerPlex 16 STR typing kits in less than 36 minutes. This rapid protocol employs commercially available polymerases and the widely used GeneAmp 9700 thermal cycler. We observed complete concordance of STR allele calls between the rapid and standard thermal cycling protocols although there was incomplete adenylation at several of the loci examined and some PCR artifacts were detected. Using less than 1 ng of template DNA and 28 cycles, STR peaks for all loci were above a 100 relative fluorescent unit (RFU) detection threshold with fully adequate inter-locus balance and heterozygote peak height ratios of greater than 0.80.