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What is RNase H2-dependent PCR?

RNase H2 enzyme is an endogenous endoribonuclease that binds to RNA-DNA duplexes, and cleaves the RNA strand, leaving a 5’ phosphate and a 3’ hydroxyl group. RNase H2 will cleave at a single ribonucleotide residue embedded within a heteroduplex; RNase H2 will not, however, cleave single-stranded RNA.

RNase H2-dependent PCR (rhPCR) uses blocked primers that cannot be extended by DNA polymerase and that contain a single ribonucleotide residue near the 3′ end. A substrate is formed for RNase H2 once the primer hybridizes to the template. RNase H2 cleaves the primer 5′ to the RNA base leaving a DNA oligonucleotide with a 3′ hydroxyl that can then be extended for DNA synthesis. The requirement for the primer to first hybridize with the target sequence before it can be extended reduces the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Thus, rhPCR can provide greater amplification specificity than standard PCR, which is especially valuable for single nucleotide polymorphism (SNP) detection.

This enzyme is available from IDT. Find out more about the IDT RNase H2 enzyme and its use in rhPCR. Contact custcare@idtdna.com with any questions you have.

Dobosy J. (2011) RNase H-dependent PCR (rhPCR): Improved specificity and single nucleotide polymorphism detection using blocked cleavable primers. BMC Biotechnology, 11(80), doi: 10.1186/1472-6750-11-80.