Figure 1

Coupled reaction scheme for PCR using blocked primers activated by cleavage with RNase H2 (rhPCR). PCR primers are designed to be incapable of extension by DNA polymerase and contain a single ribonucleotide residue near the 3'-end. Hybridization of primer to template forms a substrate for RNase H2, which will cleave the primer 5'-to the RNA base leaving a DNA oligonucleotide with a 3'-OH capable of priming DNA synthesis. The assay can be performed using either 2- or 3-step PCR with anneal/extend times as short as 30 seconds.