Caifu Chen 1,
1Integrated DNA Technologies, USA
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 multi- plex 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 map- pable reads (>97%) and on-target percentage (>97%) comparing to standard multiplex PCR.
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