In this study, a human identification (HID) RC-PCR panel was designed to target 27 identity single nucleotide polymorphisms (SNPs) generating targets only 50 base pairs in length.
Reverse Complement PCR (RC-PCR) is an innovative, one-step PCR target enrichment technology adapted for the amplification of highly degraded (fragmented) DNA. It provides simultaneous amplification and tagging of a targeted sequence construct in a single, closed-tube assay. In the current study, the complete sequencing construct was produced in a single reaction, which is essential for massively parallel sequencing (MPS) library preparation, thus reducing time and labor as well as minimizing the risk of sample carry-over or other forms of contamination. The RC-PCR system was evaluated and found to produce reliable and concordant variant calls. Also, the RC-PCR system demonstrated to have substantial sensitivity of detection with most alleles detected at 60 pg of input DNA and robustness in tolerating known PCR inhibitors. The RC-PCR system may be an effective alternative to current forensic genetic methods in the analysis of highly degraded DNA. (publisher abstract modified)