Development of a fast PCR protocol enabling rapid generation of AmpF？STR？ Identifiler？ profiles for genotyping of human DNA

Fast PCR conditions were achieved by substituting the traditional Taq polymerase for SpeedSTAR？ HS DNA polymerase which is designed for fast PCR, by upgrading to a thermal cycler with faster temperature ramping rates and by modifying cycling parameters (less time at each temperature) and adopting a two-step PCR approach.The total time required for the optimized protocol is 26 min. A total of 147 forensically relevant DNA samples were amplified using the fast PCR protocol for Identifiler. Heterozygote peak height ratios were not affected by fast PCR conditions, and full profiles were generated for single-source DNA amounts between 0.125 ng and 2.0 ng. Individual loci in profiles produced with the fast PCR protocol exhibited average n-4 stutter percentages ranging from 2.5 ± 0.9% (THO1) to 9.9 ± 2.7% (D2S1338). No increase in non-adenylation or other amplification artefacts was observed. Minor contributor alleles in two-person DNA mixtures were reliably discerned. Low level cross-reactivity (monomorphic peaks) was observed with some domestic animal DNA.The fast PCR protocol presented offers a feasible alternative to current amplification methods and could aid in reducing the overall time in STR profile production or could be incorporated into a fast STR genotyping procedure for time-sensitive situations.Novel approaches to enhance sample throughput and reduce turnaround time for the processing of casework and database samples are of high interest to the forensic community. Moreover, in some circumstances, the need for rapid human identification might be critical. Significant efforts are thus being devoted to the development of methods enabling rapid generation of STR profiles. Fast, or rapid, PCR [1-5], direct profiling circumventing DNA extraction [2,4,6], and microdevices with portable modules for on-site sample processing [3,7-9] are emerging alternatives to traditional approaches. Some protocols are already being offered to investigators for specific situations re