A New Generation of qPCR Assays Using Blocked-Cleavable Primers and RNase H2

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July 30, 2012

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  • Mark Behlke, Chief Scientific OfficerIntegrated DNA Technologies AbstractIntegrated DNA Technologies (IDT) has developed a PCR system with improved specificity called rhPCR.  The method employs modified primers that have an internal RNA residue and are 3’-end-blocked so as to be non-functional in the intact state.  Cleavage by RNase H2 removes the 3’-block and the RNA base, activating the primers.  Cleavage requires duplex formation of primer and target; mismatches near the cleavage site decrease the efficiency of cleavage by RNase H2, thereby increasing the specificity of the assay.  The new assay shows markedly reduced potential for primer-dimer formation, enabling higher levels of multiplexing in single tube assays.  rhPCR shows much higher mismatch discrimination than traditional allele-specific PCR and has utility in genotyping as well as in rare-allele detection.

    Genomics

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