English: rhPCR mechanism. rhPCR primers consist of three sections. 1) The 5’ DNA section, equivalent in length and Tm requirements to a standard PCR primer, is extended after cleavage by the RNase HII enzyme. 2) A single RNA base provides the cleavage site for the RNase HII. 3) A short 3’ extension of four or five bases followed by a blocker (usually a short, non-extendable molecule like a propanediol) prevents extension by a DNA polymerase until removal. While free in solution, these primers are not deblocked by the RNase HII enzyme, as they must be in an RNA:DNA heteroduplex with the template in order to be cleaved. They bind to the template (Figure 1), and are cleaved by the thermostable RNase HII enzyme. This removes the block, allowing for the DNA polymerase to extend off of the primers. The cycling of the PCR reaction continues the process.
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