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Session Chair: Caifu Chen, Integrated DNA Technologies, United States of America
Location:Lecture hall 14 650 participants, TUM Weihenstephan
Novel multiplex rhPCR and its applications in amplicon sequencing.
Integrated DNA Technologies, United States of America
We report here a novel multiplex PCR chemistry called rhPCR. Its primers contain a non-extendable terminal blocker and single RNA base closer to 3’. rhPCR requires both thermostable Type II RNase H (RNase H2) and DNA Polymerase for specific amplification. RNase H2 can activate rhPCR primers by target-specific cleavage of the RNA base while rhPCR primers perfectly bind to the target DNA to form a DNA duplex. Cleaved rhPCR primers can then be extended by a DNA polymerase. Combination of both 3’ blocked rhPCR primers and a highly specific mutant DNA polymerase have eliminated or greatly reduced primer dimers and non-specific amplification, resulting in higher multiplexity and better specificity than standard multiplex PCR. We have demonstrated the feasibility of the multiplex rhPCR for amplicon sequencing in multiple assay pools ranging from 96- to 1,000-plexes. Major advantages of multiplex rhPCR for amplicon sequencing include better workflow, higher mappable reads (>97%) and on-target percentage (>97%) comparing to standard multiplex PCR.
rhAmpTM A Better and Cost-Effective New SNP Genotyping Solution.
Integrated DNA Technologies, Inc., United States of America
rhAmpTM SNP Genotyping is a novel target-activated genotyping solution utilizing RNase H2-dependent PCR (rhPCR) to provide superior discrimination of single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs), and insertion/deletions (InDels). rhPCR genotyping technology combines a unique two enzyme system with DNA-RNA hybrid primers to interrogate target SNPs. Allele specific primers contain a 5’ universal tail, a single RNA base targeting the SNP, and a terminal blocking group. The 3’ blocking group is removed and primer activation 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 performance 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 universal reporter system that generates high fluorescent signal, offering increased confidence in SNP calling and the benefit of a lower cost per genotype.