Reverse complement-PCR, an innovative and effective method for multiplexing forensically relevant single nucleotide polymorphism marker systems

METHOD SUMMARY The single-step PCR library preparation method employs two reverse complement target-specific primer probes (RC probes) with a universal tail, Illumina universal i5 and i7 indexes, and sequence adapters. Region-specific primers are attached to a universal tail and contain a blocker at the 3 '-end to prevent extension. During RC-PCR, functional region-specific, tailed index PCR primers are generated and extended followed by multiplex amplification of the target regions. DNA analyses from challenging samples such as touch evidence, hairs and skeletal remains push the limits of the current forensic DNA typing technologies. Reverse complement PCR (RC-PCR) is a novel, single-step PCR target enrichment method adapted to amplify degraded DNA. The sample preparation process involves a limited number of steps, decreasing the labor required for library preparation and reducing the possibility of contamination due to less sample manipulation. These features of the RC-PCR make the technology a unique application to successfully target single nucleotide polymorphisms (SNPs) in fragmented and low copy number DNA and yield results from samples in which no or limited data are obtained with standard DNA typing methods. The developed RC-PCR short amplicon 85 SNP-plex panel is a substantial improvement over the previously reported 27-plex RC-PCR multiplex that will provide higher discrimination power for challenging DNA sample analyses.

Automated summary

The single-step PCR library preparation method utilizes RC probes, universal tails, Illumina indexes, and adapters. RC-PCR is advantageous in amplifying degraded DNA, reducing steps and contamination risk, and successfully targeting SNPs. The developed 85 SNP-plex panel of RC-PCR provides higher discrimination power than the previous 27-plex for challenging DNA sample analyses.