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

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.

Interlaboratory diagnostic validation of conformation-sensitive capillary electrophoresis for mutation scanning.

BACKGROUND: Indirect alternatives to sequencing as a method for mutation scanning are of interest to diagnostic laboratories because they have the potential for considerable savings in both time and costs. Ideally, such methods should be simple, rapid, and highly sensitive, and they should be validated formally to a very high standard. Currently, most reported methods lack one or more of these characteristics. We describe the optimization and validation of conformation-sensitive capillary electrophoresis (CSCE) for diagnostic mutation scanning. METHODS: We initially optimized the performance of CSCE with a systematic panel of plasmid-based controls. We then compared manual analysis by visual inspection with automated analysis by BioNumerics software (Applied Maths) in a blinded interlaboratory validation with 402 BRCA1 (breast cancer 1, early onset) and BRCA2 (breast cancer 1, early onset) variants previously characterized by Sanger sequencing. RESULTS: With automated analysis, we demonstrated a sensitivity of >99% (95% CI), which is indistinguishable from the sensitivity for conventional sequencing by capillary electrophoresis. The 95% CI for specificity was 90%-93%; thus, CSCE greatly reduces the number of fragments that need to be sequenced to fully characterize variants. By manual analysis, the 95% CIs for sensitivity and specificity were 98.3%-99.4% and 93.1%-95.5%, respectively. CONCLUSIONS: CSCE is amenable to a high degree of automation, and analyses can be multiplexed to increase both capacity and throughput. We conclude that once it is optimized, CSCE combined with analysis with BioNumerics software is a highly sensitive and cost-effective mutation-scanning technique suitable for routine genetic diagnostic analysis of heterozygous nucleotide substitutions, small insertions, and deletions.

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

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.