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Kristin Beltz 1, 1Integrated DNA Technologies, Inc., USA |
Abstract
rhAmpTM SNP Genotyping is a novel target-activated geno- typing solution utilizing RNase H2-dependent PCR (rhPCR) to provide superior discrimination of single nucleotide polymor- phisms (SNPs), multi-nucleotide polymorphisms (MNPs), and insertion/deletions (InDels). rhPCR genotyping technology com- bines a unique two enzyme system with DNA-RNA hybrid primers to interrogate target SNPs. Allele specific primers contain a 5′ uni- versal tail, a single RNA base targeting the SNP, and a terminal blocking group. The 3′ blocking group is removed and primer acti- vation is achieved only upon hybridization to its perfectly matched target. The thermostable RNase H2 cleaves the primer at the RNA base and releases the blocking group, allowing primer extension. PCR specificity and selectivity is improved with the use of a mutant Taq DNA polymerase and signal generation is achieved using a cost- effective universal reporter system. More than 550 human targets were selected for testing. A proprietary algorithm provided a design rate greater than 95%, and consistently high genotyping perfor- mance was achieved with greater than 90% call rate and 99.5% call accuracy on over 90% of tested assays. The rhAmpTM genotyping solution offers advanced bioinformatics driving a high assay design rate and minimal primer dimer formation, a dual enzyme master mix optimized for selectivity and stability, and an efficient univer- sal reporter system that generates high fluorescent signal, offering increased confidence in SNP calling and the benefit of a lower cost per genotype.
http://dx.doi.org/10.1016/j.bdq.2017.02.051
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