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A High-Performing and Cost-Effective SNP Genotyping Method Using rhPCR and Universal Reporters

Advances in Bioscience and Biotechnology 2018年第9期9卷 497-512页

作者： Kristin Beltz Daniel Tsang Junzhou Wang Scott Rose Yun Bao Yu Wang Katelyn Larkin Susan Rupp Daniela Schrepfer Krishnalekha Datta keith gunderson Chris Sailor Scott Hansen Joseph Dobosy Lynette Lewis Aurita Menezes Joseph Walder Mark Behlke Caifu Chen Genetic Analysis R&D Integrated DNA Technologies Coralville IA USA

We have developed a novel dual enzyme chemistry called rhAmp®SNP genotyping based on RNase H2-dependent PCR (rhPCR) that provides high signal and specificity for SNP analysis. rhAmp SNP genotyping combines a unique two-enzyme system with 3’ end blocked DNA-RNA hybrid primers to interrogate SNP loci. Activation of the blocked primers occurs upon hybridization to its perfectly matched target, which eliminates or greatly reduces primer dimers. A thermostable hot-start RNase H2 cleaves the primer immediately 5’ of the ribose sugar, releasing the blocking group and allowing primer extension. PCR specificity is further improved with the use of a mutant Taq DNA polymerase, resulting in improved allelic discrimination. Signal generation is obtained using a universal reporter system which requires only two reporter probes for any bi-allelic SNP. 1000 randomly selected SNPs were chosen to validate the 95% design rate of the design pipeline. A subsampling of 130 human SNP targets was tested and achieved a 98% call rate, and 99% call accuracy. rhAmp SNP genotyping assays are compatible with various qPCR instruments including QuantStudioTM 7 Flex, CFX384TM, IntelliQube®, and Biomark HDTM. In comparison to TaqMan®, rhAmp SNP genotyping assays show higher signal (Rn) and greater cluster separation, resulting in more reliable SNP genotyping performance. The rhAmp SNP genotyping solution is suited for high-throughput SNP genotyping applications in humans and plants.

关键词： SNP Genotyping RNase H2 rhPCR rhAmp SNP Genotyping Universal Reporter

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