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. 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 rhPCR primers (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, it is recommended that you avoid 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 avoiding having excess enzyme present for other primers in the reaction; the presence of excess enzyme will decrease specificity of cleavage.
To achieve the best performance, IDT recommends that you use blocked-cleavable primers for both forward and reverse. 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.
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 target.
Note: PCR primer pairs can be selected using the IDT PrimerQuest® program. Select qPCR 2 Primers Intercalating Dyes under Choose Your Design to get correct buffer conditions.
- 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).
- rhPrimer 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).
Figure 1. Design of rhPrimer GEN1.
Figure 2. Design of rhPrimer GEN2.
- The bases in the “disposable blocking domain” should be perfectly complementary to the target unless otherwise indicated ("M"). Mismatches placed closer to the RNA base will decrease the efficiency of cleavage by RNase H2. If you are designing primers for SNP detection, position the SNP at the RNA base, where the mismatch will have the greatest impact on RNase H2 cleavage.
- The primer domain can be longer than shown. For example, a target-specific 3’ domain can be combined with a 5’-domain that is used as a universal primer binding site to permit universal amplification after RNase H2 cleavage or subsequent capture by universal capture probes.
- 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.
Note: Primer Tm values can be calculated using the IDT OligoAnalyzer® Tool. It is important to input the buffer composition into the design tool to ensure that correct Tm values are calculated. If you are using a commercial PCR master mix and do not know the buffer composition, use the values: 50 mM KCl, 3 mM MgCl2, and 0.8 mM dNTPs, which will approximate the conditions used in most qPCR master mix recipes.