RNase H-dependent PCR

Amplification of the blocked primer is dependent on the cleavage activity of a hyperthermophilic archaeal Type II RNase H enzyme during hybridization to the complementary target sequence.

First, it has very little enzymatic activity at low temperature, enabling a “hot start PCR” without modifications to the DNA polymerase.

This allows for reduced primer dimer formation,[1] detection of alternative splicing variants,[2][3] ability to perform multiplex PCR with higher numbers of PCR primers, and the ability to detect single-nucleotide polymorphisms.

1) The 5’ DNA section, equivalent in length and melting temperature (Tm) requirements to a standard PCR primer, is extended after cleavage by the RNase HII enzyme.

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 to be cleaved.