rhPCR requires uniquely designed primers (rhPrimers) and a thermostable RNase H2 enzyme. We offer two versions of rhPrimers. rhPrimer GEN1 designs may be used for genotyping and multiplexed amplification. For more demanding studies, such as rare variant detection, rhPrimer GEN2 designs are recommended.
rhPrimer design considerations
Efficient cleavage of a blocked primer by RNase H2 requires a footprint of at least 8–10 bases upstream of the single RNA
base in the primer and 4 bases downstream of the RNA base. This footprint should be perfectly complementary to the template
intended for amplification. Mismatches can significantly reduce the efficiency of cleavage, especially when close to
the RNA cleavage site.
A blocking group (represented by x in our design nomenclature) is used either to directly block extension or to prevent replication
in subsequent cycles. Typically, a C3 spacer is used as the blocking moiety in rhPCR primers.
Two versions of rhPrimers, GEN1 and GEN2, have been developed. These have different properties and indications:
- The first generation primer (rhPrimer GEN1) is represented by DDDDDDDDrDDDDMx, where D represents a DNA base, r represents
the RNA base, M represents a mismatched DNA base, and x represents the blocker (usually a C3 Spacer). Inserting a
mismatched base before the C3 spacer to create the "Mx" combination ensures maximum effectiveness of the end block.
GEN1 primers are most appropriate for standard genotyping applications and for multiplexed amplification. This primer
design is robust and works well with low levels of RNase H2 enzyme.
- The second generation primer (rhPrimer GEN2) is represented by DDDDDDDDrDxxDM, where D represents a DNA base, r represents
the RNA base, and x represents the blocker. Inserting a mismatched DNA base at the 3′ end of the primer to create
the "DM" combination ensures maximum effectiveness of the end block. GEN2 primers are most appropriate for rare-allele
detection or for applications where extremely high fidelity of template amplification is desired. GEN2 primers may
require use of higher amounts of RNase H2 enzyme (range is 1–100X that needed for GEN1 primers; titration and optimization
need to be performed for each GEN2 primer set; for this reason we recommend use of GEN1 primers for most needs).
In general, we recommend avoiding rU as the RNA base at the cleavage site of GEN2 primers. In GEN2 format, primers containing
rU require more RNase H2 enzyme for efficient cleavage than primers containing rC, rG, or rA. If rU cannot be avoided
because of genotyping or target sequence constraints, carefully titrate the concentration of the RNase H2 enzyme to achieve
efficient cleavage of the rU primer while minimizing an excess of enzyme present for other primers in the reaction; the
presence of excess enzyme will decrease the specificity of cleavage.
To achieve the best performance, we recommend that you use blocked-cleavable primers for both forward and reverse primers.
However, using one blocked primer in conjunction with a standard primer may still improve specificity over what is typically
achievable from two standard PCR primers.
Primer design instructions
Like any other amplification reaction, good rhPCR requires use of high quality, properly designed primers.
- Select a “mature primer”, which is usually the same primer that you would normally use for standard PCR against the same
- Add the following for the rhPCR primer type:
- rhPrimer GEN1—Add an RNA base followed by 4 matching DNA bases, 1 mismatched DNA base, and the C3 blocking group
to the 3′ end of the primer (Figure 1).†
- Primer GEN2—Add an RNA base followed by a DNA base, 2 C3 blocking groups, another DNA base, and 1 mismatched DNA
base to the 3′ end of the primer (Figure 2).†
- Design the final mature primer to the same specifications you would normally use for standard PCR, ensuring that the
Tm of the primer is correct for the reaction conditions. An anneal/extend reaction temperature of 60°C is often used;
however, rhPCR is effective at a temperature range of 50–70°C.‡