A novel approach to combine qPCR and STR amplification for DNA profiling

An initial step in the development of a smart PCR machine, capable of amending the cycling parameters when amplifying STR alleles, is to monitor PCR progression in real-time. Performing qPCR allows for the real-time monitoring and recording of amplification of control loci: comprised of a small and large amplicon, a positive control, and a section of the Y chromosome. This qPCR data enables the recording of degradation and inhibition, as the fluorescence during qPCR theoretically should follow an exponential increase. Hypothetically, combining qPCR with STR amplification would allow real-time quantification of fluorescence such that the parameters of the PCR could be modified to optimise STR amplification: fluorescence below expectation would indicate a need to amend the PCR parameters to improve the DNA amplification. In this study, two different commercially available qPCR kits were combined separately with one of four different STR kits, and the resulting STR profile quality was recorded. Controls were performed by amplifying the same quantity of DNA template for each of the four STR kits, with all standard single and combined amplifications performed five times, resulting in 60 amplifications in total. No significant decrease in profile quality or likelihood ratios were recorded for any of the combinations. There were no adverse effects on the STR amplification when performed on a real-time PCR machine, despite two different enzymes and the presence of additional primers requiring differing temperatures to bind. These data are needed as the first step towards a smart PCR machine that can adjust cycling parameters in real-time.