The effects of extra PCR cycles when amplifying skeletal samples with the GlobalFiler^® PCR Amplification Kit

When samples with low amounts of DNA are amplified using short tandem repeats (STRs), stochastic effects such as allele and locus dropout or drop-in, allele imbalance, and increased stutter often occur making data interpretation more difficult. The most common approach to improving STR results from low template samples is to increase the number of PCR cycles. Although more alleles may be recovered, stochastic effects may be exaggerated resulting in more complicated STR profiles. This work reports the effect of additional PCR cycles (29 vs. 30, 31, and 32) on STR success from environmentally challenged bone and tooth samples using the GlobalFiler^® DNA Amplification Kit (Thermo Fisher Scientific). In addition, we compared the efficiency of two DNA extraction kits for skeletal samples: QIAamp^® DNA Investigator (QIAGEN) and PrepFiler^® BTA™ Forensic DNA Extraction (Thermo Fisher Scientific) kits. Results showed that more DNA was recovered from samples using the PrepFiler^® BTA™ kit; but regardless of the extraction method, the number of alleles detected and the peak heights both increased with an increase in PCR cycle number. Although more alleles were reported in almost all samples, the most notable improvement was observed in samples with the DNA template < 120 pg. A general increase in the number of PCR artifacts was detected in STR profiles generated using 30-32 cycles. Overall, this study provides supporting evidence that STR profile completeness and quality may be improved when low template skeletal samples are amplified with extra PCR cycles (up to 32 cycles) using the GlobalFiler^® DNA Amplification Kit.