Reverse Complement PCR: A novel one-step PCR system for typing highly degraded DNA for human identification

Area of Science:

• Molecular Biology
• Forensic Genetics
• Biotechnology

Background:

• Analysis of highly degraded DNA presents significant challenges in forensic science.
• Existing methods for DNA amplification and tagging can be time-consuming and prone to contamination.
• Need for efficient and reliable techniques for human identification from fragmented DNA samples.

Purpose of the Study:

• To introduce and evaluate Reverse Complement PCR (RC-PCR) as a one-step target enrichment technology.
• To assess the utility of RC-PCR for human identification (HID) using a panel of single nucleotide polymorphisms (SNPs).
• To determine the efficiency, sensitivity, and robustness of RC-PCR for degraded DNA analysis.

Main Methods:

• Development of a human identification RC-PCR panel targeting 27 identity SNPs with short amplicons (50 bp).
• Simultaneous amplification and tagging of targeted sequences in a single, closed-tube reaction.
• Evaluation of variant calls, sensitivity (down to 60 pg DNA), and tolerance to PCR inhibitors.

Main Results:

• RC-PCR successfully generated complete sequencing constructs for massively parallel sequencing (MPS) library preparation.
• The system produced reliable and concordant variant calls.
• High sensitivity was demonstrated, with most alleles detected at 60 pg input DNA, and robustness against PCR inhibitors was observed.

Conclusions:

• RC-PCR is an innovative, efficient, and reliable one-step method for amplifying and tagging degraded DNA.
• The technology reduces time, labor, and contamination risks in genetic analysis.
• RC-PCR shows promise as an effective alternative to current forensic genetic methods for analyzing highly degraded DNA samples.