This study demonstrated a rapid cycling protocol that amplified 15 STR loci and the sex-typing marker amelogenin from the Identifiler STR typing kit in less than 36 minutes, using commercially available polymerases and the widely used GeneAmp 9700 thermal cycler.
Even if some artifacts arise during rapid PCR protocols, such as in complete adenylation or a few nonspecific products, the STR profile information can still be valuable for general screening and informational purposes. In simple screening situations, there should be sufficient quantities of single-source, high quality DNA available. Until recently, success with amplifying large multiplexes in less than 1 hour has been limited. The STR performance parameters of locus-to-locus balance, locus or allelic drop out, incomplete adenylation, low peak height ratio, and general robustness must be evaluated when developing a rapid PCR protocol. Some of these problems were addressed with additional enzyme or altering cycling times; however, others are specific to a locus, primer pair sequence, or primer pair concentration present in a commercial primer mix. From the results presented with a commercial kit, there is now a basis for understanding the limitations of using a “fixed” (primer sequences and concentration) primer mix. It also allows for directing the future focus on specific aspects of primer design in order to resolve these issues when developing new rapid multiplex PCR assays. Additional experiments to perform include evaluating more rapid thermal cycling instrumentation capable of faster temperature ramp rates, investigating the impact of further variations in PCR volumes, further optimization of annealing temperatures, and determining levels of sensitivity. The authors also plan to examine additional non-kit STR loci where primer concentrations can be modified in order to adjust locus-to-locus balance and primers ends can be modified to aid adenylation. 1 table, 1 figure, and 14 references