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Reverse Complement-PCR, An Innovative and Effective Method for Multiplexing Forensically Relevant Single Nucleotide Polymorphism Marker Systems

NCJ Number
Date Published
August 2021

Since DNA analyses from challenging samples such as touch evidence, hairs, and skeletal remains push the limits of the current forensic DNA typing technologies, this article proposes and describes the use of reverse complement PCR (RC-PCR), 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 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. The single-step PCR library preparation method uses 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. (publisher abstract modified)

Date Published: August 1, 2021