Abstract

Target-activated RNase H2 PCR for Qualitative and Quantitative SNP Genotyping

Caifu Chen, Senior Vice President, Integrated DNA Technologies

We report here a new genotyping method called target-activated RNase H2 PCR (rhPCR) for both qualitative and quantitative genotyping of SNPs. This method involves cleavage and activation of blocked rhPCR primers, allele-specific PCR with universal tails, and generation of fluorescence signals using 2, 3 or 4 universal reporters which, respectively, detect di-, tri- or tetra-alleles of a SNP. A rhPCR primer has a doped RNA base and a blocker at 3’ which is not extendable by Taq polymerase. The blocked rhPCR primer is activated only by binding to the perfectly matched target and subsequent cleavage of its RNA base by RNase H2. Therefore, primer dimers are eliminated or significantly reduced during rhPCR. Elimination of primer dimers is believed to be the key to robust universal reporter-based genotyping assays. A mutant Taq polymerase is used to increase the PCR specificity and selectivity. A total of 300 human SNPs have been analyzed with 48 Coriell genomic DNA samples with 95% design rate, >90% assay pass rate, >99% call rate and 99.9% accuracy. Use of this chemistry for quantitative genotyping to determine both genotypes and their copy numbers at end-point PCR will also be discussed.