RNase H2 PCR—A New Technology to Reduce Primer Dimers and Increase Genotyping Accuracy

RNase H–Dependent PCR Primers and Enzyme

Amplification technology developed by IDT that facilitates SNP genotyping applications, high levels of multiplexed amplification, and detection of rare alleles. 

RNase H-dependent PCR (rhPCR) relies on cleavage of a 3’-blocked primer by RNase H2 to effect primer activation before target amplification. The cleaved, unblocked primer is then available to support extension and replication in the polymerase chain reaction (PCR). Cleavage occurs at the 5’ end of a single RNA base that is incorporated near the 3’ end of the primer, leaving a 3’-hydroxyl on the terminal DNA base. A blocking moiety included downstream of the RNA base can block either direct extension of the primer or replication by the polymerase during subsequent cycles of PCR. The blocked primer can only be cleaved when bound to its complementary target sequence and at elevated temperature, which strongly reduces the formation of primer-dimers or misprimed species that can result in unwanted amplification products.

Blocked-cleavable rhPCR primers (rhPrimers) and the thermostable RNase H2 enzyme required for rhPCR are available from IDT.

Note: Primer design information is now located in the Overview tab.

rhPCR Primers

Prices for primers up to 60 nt are shown. Primer lengths affect yield guarantees, which will be provided in customer quotes.

Primer TypeTerminal BasesSynthesis
rhPrimer GEN1rDDDDMx100 nmole$25.00 USD$60.00 USD
rhPrimer GEN2rDxxDM100 nmole$35.00 USD$60.00 USD

* Standard desalting purification 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.


D = DNA base; match to target
r = RNA base; match to target
M = DNA base; mismatch to target
x = C3 spacer

Create your own custom rhPCR Primer designs, or contact IDT Technical Support for design assistance.

To order rhPCR Primers, complete the Order Form and email to custcare@idtdna.com.

RNase H2 Enzyme and Buffer

RNase H2 enzyme for use with rhPCR primers is available at two concentrations, 20 U/µL and 2 U/µL. RNase H2 is functional in most PCR buffers and can be added directly to the PCR master mix. The enzyme must be diluted before use. Please use the Enzyme Dilution Buffer that is supplied with the enzyme.

One unit is defined as the amount of enzyme necessary to nick 1 nmol of synthetic, single-rC–containing duplex substrate per minute at 70°C in 10 mM Tris, 50 mM NaCl, 0.01% Triton X-100, 10 µg/mL BSA, and 4 mM MgCl2. Depending on the application, enzyme usage ranges from 2–200 mU per reaction (see Protocol).

The RNase H2 Enzyme Kit contains RNase H2 Enzyme (50 U at 2 U/µL) and RNase H2 Enzyme Dilution Buffer (2 mL).

DescriptionPart #Pricing
RNase H2 Enzyme Kit11-02-12-01$200.00 USD
RNase H2 Enzyme Small - 50 U at 2U/uL11-03-02-02$200.00 USD
RNase H2 Enzyme Large - 500 U at 20U/uL11-03-02-03$2,000.00 USD
2 mL RNase H2 Enzyme Dilution Buffer11-01-02-12$4.00 USD
10 x 2 mL RNase H2 Enzyme Dilution Buffer11-01-03-08$35.00 USD


How rhPCR Works

High specificity is often achieved with the polymerase chain reaction (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 blocked primers that contain a single ribonucleotide residue. The blocked primers are activated when cleaved by the RNase H2 enzyme. Cleavage occurs at the 5’ end of  the RNA base after primer hybridization to the target DNA. Because the primers must hybridize to the target sequence before they are cleaved, they are unable to form primer dimers and the requirement for high target complementarity reduces amplification of closely related sequences (Figure 1). rhPCR is more sensitive than allele-specific PCR for detection of single-nucleotide polymorphisms (SNPs). Superior allele discrimination is achieved when the mismatched base is positioned at the RNA:DNA base pair [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 active). 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. [1].

Figure 1. Schematic Representation of rhPCR


  1. Dobosy JR, Rose SD, Beltz KR, Rupp SM, Powers KM, Behlke MA, and Walder JA (2011) RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers. BMC Biotechnol, 11:80.


rhPCR Primer 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. 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.

Primer Design

Like any other amplification reaction, good rhPCR requires use of high quality, properly designed primers.

  1. Select a ‘mature primer,’ which is usually the same primer that you would normally use for standard PCR against the same target.

  2. 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.

  3. Add the following for the rhPCR primer type:
    1. 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).
    2. 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).

  4. Figure 1. Design of rhPrimer GEN1.

    Figure 2. Design of rhPrimer GEN2.


    1. 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.
    2. 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.

  5. 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.

  6. 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.

Product Documentation

Certificates of Analysis

RNase H2 Enzyme Small

RNase H2 Enzyme Large

RNase H2 Dilution Buffer

Safety Data Sheets (SDSs)

Pyrococcus abyssi RNase H2 Enzyme



Pyrococcus abyssi RNase H2 can be incubated at 95°C for >45 minutes with little loss of activity and therefore, can 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.


Compatibility with PCR Temperatures

Pyrococcus abyssi RNase H2 is active at temperatures that are compatible with PCR conditions and inactive at room temperature. 

  • P. abyssi RNase H2 can be used to create “hot start” PCR conditions without the need for a modified “hot start” polymerase, because RNase H2 is essentially inactive at room temperature.
  • You can optimize reaction conditions (e.g., increase the amount of RNase H2 per reaction), if your PCR cycling conditions require annealing/extension temperatures between 50–60°C (data not shown).
  • P. abyssi RNase H2 activity reaches a plateau between 70–95°C (data not shown).
  • Compared to other RNases, P. abyssi RNase H2 is relatively safe to have in laboratories that work with RNA, because this RNase H2 requires elevated temperatures, Mg++, detergents, and duplexed RNA/DNA substrate for activity.
RNaseH2, Activity Vs. Temperature

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).


Compatibility with PCR Mg++ Concentrations

Pyrococcus abyssi RNase H2 is active across a broad range of Mg++ concentrations that are compatible with PCR conditions (typically 3 mM).

RNaseH2 Activity vs. [Mg++]

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.


Detergent Requirements

Detergent is required for robust Pyrococcus abyssi RNase H2 activity.

  • The IDT dilution and storage buffer for RNase H2 contains 0.1% Triton™ X-100.
RNaseH2, Detergent Requirement

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.