How rhPCR works
High specificity is often achieved with PCR; however, sometimes it is necessary to position primers at suboptimal locations in the target. This can result in the formation of primer-dimers and/or undesired amplification of homologous sequences.
IDT scientists have developed RNase H2–dependent PCR, a method for increasing PCR specificity and eliminating primer-dimers by using RNase H2 from Pyrococcus abyssi (P.a.) and DNA primers that contain a single ribonucleotide residue and a 3′ blocking moiety. The blocked primers are activated when cleaved by the RNase H2 enzyme. Cleavage occurs on the 5′ side of the RNA base after primer hybridization to the target DNA. Because the primers can only be cleaved after they hybridize to the perfectly matched target sequence, primer-dimers are reduced. The requirement for high target complementarity reduces amplification of closely related sequences (Figure 1).
Pyrococcus abyssi is an extreme thermophile, so the P.a. RNase H2 enzyme has optimal activity in range of 70–75°C and is functional in rhPCR between 50°C and 75°C. P.a. RNase H2 has very low activity at room temperature (~1000X less activity). Therefore, use of this enzyme to perform primer activation confers a "hot start" character to the PCR. P.a. RNase H2 is functional in most PCR buffers and can be added directly to the PCR master mix. The enzyme functions in real time, making the method a transparent change to standard PCR with primer cleavage occurring in the background during each anneal/extend cycle. .