Development of the AmpFISTR SEfiler PCR amplification kit: a new multiplex containing the highly discriminating...

The AmpFISTR SEfiler kit co-amplifies 11 short tandem repeat loci including SE33 in a single multiplex. After establishing the optimum in primer titration studies, the primer concentrations of all loci in the multiplex were chosen such that the heterozygote peak height ratios of each of the loci were balanced. The combined primer set was then tested to determine the robustness of the multiplex under various conditions. Different MgCl(2) concentrations were evaluated to establish the optimum concentration for the multiplex. The amplification of the various loci in the multiplex was tested at several annealing temperatures (55-63 degrees C). Additionally, DNA from primates, non-primates and microorganisms were amplified to investigate the specificity of the kit. The stability of the AmpFISTR SEfiler kit was determined by addition of hematin, to simulate inhibition, and the use of degraded DNA. Population studies revealed a probability of identity of 6.47x10(-15) for African Americans and 7.46x10(-14) for US Caucasians. To assess the ability of the multiplex to analyze forensic samples, testing on blood, oral swabs and mixtures was performed. Based on the various studies, it was determined that the AmpFISTR SEfiler PCR amplification kit can be used to successfully analyze a variety of forensic, databasing and paternity samples.