Rapid PCR protocols for forensic DNA typing on six thermal cycling platforms

Rapid PCR protocols for the amplification of typing STR multiplexes were evaluated on six different thermal cyclers. Through the use of a faster DNA polymerase coupled with the use of rapid thermal cyclers the amplification cycling times were reduced down to as little as 14 min using PCR primers from the commercially available multiplex STR typing kit Identifiler. Previously described two-step and three-step thermal cycling protocols were evaluated for the six thermal cyclers on 95 unique single-source DNA extracts. CE characterization of the PCR products indicates good peak balance between loci (median values greater than 0.84), and N minus four stutter ratios on averages were 30 to 40% higher than for standard Identifiler PCR conditions. Nonspecific amplification artifacts were observed, but were not observed to migrate within the allele calling bins. With the exception of one locus (D18S51) in a single sample, genotyping results were concordant with manufacturer's recommended amplification conditions utilizing standard thermal cycling procedures. Assay conditions were robust enough to routinely amplify 250 to 500 pg of template DNA. This work describes the protocols for the rapid PCR amplification of STR multiplexes on various PCR thermal cyclers with the future intent to support validation for typing single-source samples in a database laboratory.