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

Rachel E Kieser; Magdalena M Buś; Jonathan L King; Walter van der Vliet; Joop Theelen; Bruce Budowle

doi:10.1016/j.fsigen.2019.102201

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

Forensic Sci Int Genet. 2020 Jan:44:102201. doi: 10.1016/j.fsigen.2019.102201. Epub 2019 Nov 6.

Authors

Rachel E Kieser¹, Magdalena M Buś², Jonathan L King³, Walter van der Vliet⁴, Joop Theelen⁴, Bruce Budowle²

Affiliations

¹ Center for Human Identification, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA; Department of Microbiology, Immunology, and Genetics, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA. Electronic address: Rachel.Kieser@my.unthsc.edu.
² Center for Human Identification, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA; Department of Microbiology, Immunology, and Genetics, Graduate School of Biomedical Sciences, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA.
³ Center for Human Identification, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA.
⁴ NimaGen B.V., Lagelandseweg 56, 6545 CG Nijmegen, the Netherlands.

PMID: 31786458
DOI: 10.1016/j.fsigen.2019.102201

Abstract

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. A human identification (HID) RC-PCR panel was designed targeting 27 identity single nucleotide polymorphisms (SNPs) generating targets only 50 base pairs in length. In a single reaction, the complete sequencing construct is produced 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 a majority of 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.

Keywords: Degraded DNA; Identity SNPs; RC-PCR; Reverse Complement PCR; SNPs.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Alleles
DNA Degradation, Necrotic*
DNA Fingerprinting / methods*
DNA Fragmentation*
Forensic Genetics / methods
High-Throughput Nucleotide Sequencing
Humans
Polymerase Chain Reaction / methods*
Polymorphism, Single Nucleotide