PCR with RNase H (rhAmp PCR)

Reduce primer dimers and other undesirable amplification products with state-of-the-art PCR technology

What is rhAmp PCR?

rhAmp PCR is RNase H-dependent PCR (rhPCR), a nucleic acid amplification method that provides increased target specificity over traditional PCR. Compared to traditional PCR, rhAmp PCR requires an additional enzyme, RNase H2, and uses blocked primers (rhPrimers or rhAmp Primers) in place of conventional PCR primers.

How does it work?

rhAmp technology relies heavily on the unique design of rhPrimers and the inherent characteristics of RNase H2. As opposed to DNA-only primers used in traditional PCR and qPCR, rhPrimers contain a single RNA base and a 3′ blocking group that must be removed by RNase H2 before extension by DNA polymerase can occur (Figure 1). The added specificity achieved by this mechanism consistently yields more amplification of the fragments you are targeting, and less off-target amplification (Figure 2).

Who should use it?

The primer-dimer reducing abilities of rhAmp technology make it ideal for demanding genomics applications like multiplexed amplicon sequencing and SNP genotyping. In addition, if you are performing studies involving rare variant detection, splice variant discovery, or microbial identification, consider using rhAmp PCR to create your own PCR assay. Its added specificity can offer both efficiency and accuracy improvements to regular PCR when undesirable amplification artifacts are a consistent problem.

Figure 1. Schematic representation of rhAmp PCR. DNA primers containing a single RNA residue and a 3′ blocking moiety (rhPrimers) are activated when cleaved by 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 and other undesirable amplification products are reduced.

Figure 2. rhAmp PCR primers virtually eliminate primer dimers and nonspecific amplification artifacts in multiplex applications. In multiplex PCR amplification of 96 targets from human genomic DNA (NA12878, Coriell Institute), two sets of multiplex primers for the 96 assays (192 individual primers) were synthesized either as standard PCR primers or as rhAmp PCR primers (rhPrimers). The results show tight control of primer-dimer artifacts, even in highly multiplexed assays. Total reaction volumes were 50 µL, with 10 ng of genomic DNA template or no template as controls. RNase H2 was present in all reactions but has no functional role in PCRs with standard primers.

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