TITLE:
A High-Performing and Cost-Effective SNP Genotyping Method Using rhPCR and Universal Reporters
AUTHORS:
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
KEYWORDS:
SNP Genotyping, RNase H2, rhPCR, rhAmp SNP Genotyping, Universal Reporter
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.9 No.9,
September
29,
2018
ABSTRACT: 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.