A High-Performing and Cost-Effective SNP Genotyping Method Using rhPCR and Universal Reporters
A High-Performing and Cost-Effective SNP Genotyping Method Using rhPCR and Universal Reporters作者机构：Genetic Analysis R&D Integrated DNA Technologies Coralville IA USA
出 版 物：《Advances in Bioscience and Biotechnology》 (生命科学与技术进展（英文）)
年 卷 期：2018年第9卷第9期
页 面：497-512页
学科分类：1002[医学-临床医学] 100214[医学-肿瘤学] 10[医学]
主 题：SNP Genotyping RNase H2 rhPCR rhAmp SNP Genotyping Universal Reporter
摘 要：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.
维普期刊数据库