Precise amplification of target sequences in applications such as rare variant detection, splice variant discovery, and microbial identification
rhPCR is a novel nucleic acid amplification technology that provides improved accuracy over traditional PCR. rhPCR primers (rhPrimers), unique primers that contain RNA bases, are used in conjunction with the thermostable RNase H2 enzyme to perform rhPCR.
In addition to your choice of Taq DNA polymerase and other general PCR reagents, rhPCR technology requires the following components:
Key: D = DNA base, match to target; r = RNA base, match to target; M = DNA base, mismatch to target; x = C3 space
* Standard desalting is sufficient for most situations and is included in the price of each primer. For experiments with long primers or requirements for above average purity, RNase-free HPLC purification is available for an additional fee.
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.
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:
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.
Like any other amplification reaction, good rhPCR requires use of high quality, properly designed primers.
* PCR primer pairs can be selected using the PrimerQuest® program. Select qPCR 2 Primers Intercalating Dyes under Choose Your Design to get correct buffer conditions.
† 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.
‡ Primer Tm values can be calculated using the 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.
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. .
Pyrococcus abyssi RNase H2 can be incubated at 95°C for >45 minutes with little loss of activity (Figure 2) and, therefore, can survive PCR conditions.
Figure 2. Thermostability of P. abyssi RNase H2 allows this enzyme to survive PCR conditions. P. abyssi RNase H2 was preincubated at 95°C for 0–90 min in buffer (10 mM Tris HCl, pH 8.0; 50 mM NaCl; 0.01% Triton X-100; 10 µg/mL BSA; and 4 mM MgCl2). Radiolabeled substrate (2 pmol) was added to heat-treated RNase H2 (100 µU), and enzyme activity was assessed by measuring substrate cleavage after incubation at 70°C for 20 min.
Pyrococcus abyssi RNase H2 is active at temperatures that are compatible with PCR conditions and inactive at room temperature (Figure 3).
Figure 3. P. abyssi RNase H2 is active at temperatures compatible with PCR and inactive at room temperature (20–25°C). Enzyme activity of P. abyssi RNase H2 (250 µU) was assessed by measuring cleavage of a radiolabeled substrate (2 pmol) after incubation at varying temperatures for 20 min in buffer (10 mM Tris HCl, pH 8.0; 50 mM NaCl; 0.01% Triton X-100; 10 µg/mL BSA; and 4 mM MgCl2).
Pyrococcus abyssi RNase H2 is active across a broad range of Mg2+ concentrations (Figure 4) that are compatible with PCR conditions (typically 3 mM).
Figure 4. P. abyssi is active at Mg2+ concentrations compatible with PCR. Enzyme activity of P. abyssi RNase H2 (250 µU) was assessed by measuring cleavage of a radiolabeled substrate (2 pmol) after incubation at 70°C for 20 min in buffer (10 mM Tris HCl, pH 8.0; 50 mM NaCl; 0.01% Triton X-100; and 10 µg/mL BSA), containing 0–8 mM MgCl2.
The highest enzyme activity is observed with Triton™ X-100 and Tween® 20 (Figure 5).
Figure 5. Robust P. abyssi RNase H2 activity requires detergent. Enzyme activity of P. abyssi RNase H2 (100 µU) was assessed by measuring cleavage of a radiolabeled substrate (2 pmol) after incubation at 70°C for 30 min in buffer (10 mM Tris HCl, pH 8.0; 50 mM NaCl; 10 µg/mL BSA; and 4 mM MgCl2), containing varying amounts of different detergents.
rhPCR uses activation of RNA-DNA hybrid primers by the RNase H2 enzyme from Pyrococcus abyssi (see Technology overview section). The primers are only activated upon precise annealing to the correct target, and this RNase H2 activation mechanism nearly eliminates primer-dimers and other amplification artifacts that can affect experimental results. Figure 1 shows how effectively RNase H2 activation for elimates primer-dimer artifacts, even when 96 primer pairs are multiplexed in a single PCR.
Figure 1. rhPCR 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 RNase H2–activated, rhPCR primer pairs. 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.