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.

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
Scale
PriceRNase-Free
HPLC*
rhPrimer GEN1rDDDDMx100 nmole$25.00 USD$60.00 USD
rhPrimer GEN2rDxxD100 nmole$35.00 USD$60.00 USD

* The fee for RNase-Free HPLC purification is in addition to the base price for each primer.

Key:

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.

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

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


References

  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.

    Notes:

    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.


Protocol

The procedure for rhPCR is similar to standard qPCR, but requires blocked-cleavable primers (rhPrimers) and the addition of RNase H2 enzyme to the master mix. Pyrococcus abyssi (P.a.) RNase H2 is thermostable and will continue to function throughout PCR cycling.

P.a. RNase H2 is available at two concentrations, 20 U/µL and 2 U/µL. The enzyme must be diluted before use. Please use the Enzyme Dilution Buffer that is supplied with the enzyme.

  • Use 2–10 mU RNase H2 for 10 µL reactions that contain rhPrimers GEN1. For larger reactions, the amount of enzyme used should be scaled up proportionally (i.e., use 5–25 mU RNase H2 for 25 µL reactions. Start with 5 mU/10 µL and titrate the enzyme up or down so that reaction efficiency is similar to control reactions that have been set up using unmodified primers. Using insufficient enzyme will lower reaction efficiency and will require additional PCR cycles. Excess enzyme will decrease specificity, removing the benefit of performing rhPCR.
  • Use 5–200 mU RNase H2 per 10 µL reaction mix for reactions that contain rhPrimers GEN2. If one of the primers contains a rU residue at the cleavage site, it might be necessary to use more enzyme than for primers that contain rA, rG, or rC. As with GEN1 primers, titrate the amount of RNase H2 needed.
  • Use more RNase H2 for multiplex reactions than for singleplex reactions. The precise amount needed for a multiplex reaction varies with the number of amplicons being detected, primer concentration, and buffer composition. We recommend that you optimize the reaction by testing a
    variety of enzyme concentrations.
  • Ensure a minimum final concentration of 0.01% Triton X100 (or equivalent non-ionic detergent) in the reaction. The enzyme dilution buffer provided with RNase H2 contains 0.1% Triton X100. Therefore, if a 10X stock enzyme solution is used, simply add the enzyme in a 1:10 ratio into the reaction mix to achieve the correct Triton X100 concentration. The reaction will not be affected if detergent levels are 2–3X above the recommended minimum, but reaction efficiency decreases if detergent drops below this level.

IDT scientists have tested compatibility of P.a. RNase H2 and rhPCR with many commercial PCR master mixes using manufacturers' recommended cycling conditions and IDT "standard" cycling conditions* (Tables 1 and 2). Master mixes containing SYBR® Green and other intercalating dyes (Tables 3 and 4) and commercial DNA polymerases (Tables 5 and 6) have also been tested using IDT standard cycling conditions. Most master mixes and polymerases perform well; however, empirical testing of each mix or polymerase is required because buffer composition affects reaction performance. In general, high fidelity polymerases with 3’-exonuclease activity perform poorly with rhPrimers GEN1 (rDDDDMx) because the exonuclease function removes the 3’ blocking group, allowing amplification to occur in the absence of RNase H2 and removing the benefits of using blocked-cleavable primers. However, the 3' block for rhPrimers GEN2 (rDxxD) is more stable, so these are compatible with high fidelity 3'-exo polymerases.

* IDT standard cycling conditions: 3 min, 95°C; 45 x (10 sec, 95°C; 30 sec, 60°C)


Table 1. Compatibility of rhPrimers GEN1 (rDDDDMx) With Commercial Master Mixes. 

Master Mix
Amount of RNase H2 Required per 10 µL Reaction (mU)
Manufacturer's Cycling IDT Standard Cycling*
Applied Biosystems TaqMan® Fast Advanced 5 5
Applied Biosystems TaqMan Gene Expression 1.3 2.6
Bio-Rad iTaq™ DNA Polymerase 1.3 2.6
Bio-Rad iQ™ Multiplex Powermix 2.6 2.6
Bio-Rad SsoFast™ Probes Supermix 5 2.6
Invitrogen EXPRESS qPCR Supermix 1.3 2.6
KAPA Probe Fast qPCR 5 2.6
PCR Biosystems qPCRBIO Probe Mix Lo-ROX 5 2.6
Quanta PerfeCTa® Multiplex qPCR SuperMix 2.6 1.3
Qiagen Multiplex PCR Plus Kit Not recommended Not recommended
Qiagen QuantiTect® Multiplex PCR Kit 2.6 2.6
Roche FastStart TaqMan Probe Master Not recommended Not recommended

* IDT standard cycling conditions: 3 min, 95°C; 45 x (10 sec, 95°C; 30 sec, 60°C)



Table 2. Compatibility of rhPrimers GEN2 (rDxxD) With Commercial Master Mixes.

Master Mix
Amount of RNase H2 Required per 10 µL Reaction (mU)
Manufacturer's Cycling IDT Standard Cycling
Applied Biosystems TaqMan® Gene Expression 100 150
Bio-Rad iTaq™ DNA Polymerase 200 200
Bio-Rad iQ™ Multiplex Powermix 150 150
Bio-Rad SsoFast™ Probes Supermix 400 150
Invitrogen EXPRESS qPCR Supermix 100 100
KAPA Probe Fast qPCR 100 150
PCR Biosystems qPCRBIO Probe Mix Lo-Rox 100 100
Quanta PerfeCTa® Multiplex qPCR SuperMix 100 100
Qiagen Multiplex PCR Plus Kit Not recommended Not recommended
Qiagen QuantiTect® Multiplex PCR Kit 200 200
Note: This survey was performed using a forward primer with “rUDxxD” design and an unmodified  reverse primer. If neither primer contains a rU residue, the amount of RNase H2 required to achieve peak cycling efficiency will be lower. 

† IDT standard cycling conditions: 3 min, 95°C; 45 x (10 sec, 95°C; 30 sec, 60°C)



Table 3. Compatibility of rhPrimers GEN1 (rDDDDMx) With SYBR® Green Dye–Based and Similar Master Mixes Using IDT Standard Cycling Conditions*.

Master Mix Amount of RNase H2 Required per 10 µL Reaction (mU)
Agilent Brilliant SYBR® Green 2.6
Agilent Brilliant II SYBR® Green 2.6
Applied Biosystems SYBR® Green PCR 2.6
Bio-Rad iQ™ SYBR Green Supermix 2.6
Bio-Rad SsoAdvanced™ SYBR® Green 50
Bio-Rad SsoFast™ Eva Green 50
Bio-Rad SYBR® Fast qPCR 5
Biotium Fast EvaGreen® qPCR
2.6
Invitrogen Platinum SYBR® Green qPCR Supermix-UDG
2.6
Promega GoTaq® Green
2.6
Qiagen HotStarTaq® Plus + Invitrogen SYBR® GreenER™
2.6
Qiagen QuantiTect® SYBR® Green
2.6
Roche LightCycler® 480 SYBR® Green I 2.6

* IDT standard cycling conditions: 3 min, 95°C; 45 x (10 sec, 95°C; 30 sec, 60°C)



Table 4. Compatibility of rhPrimers GEN2 (rDxxD) With SYBR® Green Dye–Based and Similar Master Mixes Using IDT Standard Cycling Conditions*.

Master Mix Amount of RNase H2 Required per 10 µL Reaction (mU)
Agilent Brilliant SYBR® Green 100
Agilent Brilliant II SYBR® Green 50
Applied Biosystems SYBR® Green PCR 100
Bio-Rad iQ™ SYBR® Green Supermix 200
Biotium Fast EvaGreen® qPCR
200
Invitrogen Platinum SYBR® Green qPCR Supermix-UDG
50
Promega GoTaq® Green
200
Qiagen HotStarTaq® Plus + Invitrogen SYBR® GreenER™
50
Qiagen QuantiTect® SYBR® Green PCR Kit
Not recommended
Roche LightCycler® 480 SYBR® Green I 100

* IDT standard cycling conditions: 3 min, 95°C; 45 x (10 sec, 95°C; 30 sec, 60°C)




Table 5. Compatibility of rhPrimers GEN2 (rDxxD) With High Fidelity DNA Polymerases Using IDT Standard Cycling Conditions*.

DNA Polymerase Amount of RNase H2 Required per 10 µL Reaction (mU)
Agilent Herculase II Fusion 400
Agilent PfuUltra II HiFi Hotstart 400
Biolilne MyFi™ 50
Bioline RANGER 50
Bioline VELOCITY™ 200
Enzymatics VeraSeq™ 2.0 High-Fidelity 200
KAPA Biosystems KAPA HiFi™ Hotstart  Not recommended
NEB Q5® Hot Start High-Fidelity
Not recommended
PCR Biosystems PCRBIO HiFi Hotstart
200
Roche FastStart HiFi  100
Syzygy (Empirical Bioscience) SyFi
Not recommended
Thermo Scientific Phire Hot Start II
400
Thermo Scientific Phusion HiFi Hotstart 200

* IDT standard cycling conditions: 3 min, 95°C; 45 x (10 sec, 95°C; 30 sec, 60°C)




Table 6. Compatibility of rhPrimers GEN1 (rDDDDMx) With Various DNA Polymerases and IDT Standard Cycling Conditions.

DNA Polymerase* Amount of RNase H2 Required per 10 µL Reaction (mU)
Enzymatics Taq-B 1.3
Bio-Rad iTaq™ 1.3
Promega Tfl 2.6
Clontech Titanium® Taq 1.3
Qiagen HotStarTaq® 2.6
Applied Biosystems Amplitaq Gold® 2.6
Applied Biosystems Amplitaq® 2.6
Thermo Scientific DyNAzyme™ II Hot Start 1.3
Roche FastStart Taq 1.3
NEB Deep VentR™ (exo-) 2.6
NEB VentR® (exo-) 2.6
Bioline Immolase™ 10
Millipore KOD Hot Start 10
NEB Tli Inconsistent results
Agilent PfuUltra High-Fidelity No amplificiation
Thermo Scientific Phusion® High-Fidelity Blocked primers are cleaved without RNase H2
Thermo Scientific Phire® Hot Start >10 mU RNase H2 required
NEB Deep VentR™ Blocked primers are cleaved without RNase H2
NEB VentR® Blocked primers are cleaved without RNase H2
NEB 9°Nm™ Unusual amplification curves; not recommended
* Note: The buffer recommended for each polymerase was used. Some polymerases may work with rhPCR if the buffer composition is modified. However, because most polymerase vendors do not provide buffer composition, it is difficult to adjust individual components.

† IDT standard cycling conditions: 3 min, 95°C; 45 x (10 sec, 95°C; 30 sec, 60°C)




Example of a Homemade rhPCR Mix for rhPrimers GEN1

Component Final Concentration
Tris pH 8.4 20 mM
KCl 50 mM
MgCl2 3.0 mM
dNTPs 0.8 mM (0.2 mM each)
Triton X100 0.01%
Forward primer 200 nM
Reverse primer 200 nM
Target DNA (variable)
Taq polymerase 0.5 U
P.a. RNase H2 0.5 mU/µL (1 µL of a 5 mU/µL stock)
Final volume 10 µL

PCR cycling conditions:

  • 95°C soak, 2–10 min (to activate the hot start polymerase)
  • [95°C, 10 sec; 60°C, 30–60 sec] x 40 cycles

Notes:

  1. IDT recommends use of a 2-step PCR cycle. During PCR, the blocked-cleavable primers anneal to the target and are activated (cleaved by RNase H2) during the anneal phase of the reaction. In a 2-step PCR cycle, the anneal phase also serves as the polymerase extend phase, so this phase is longer and allows for the highest amount of primer activation. In a 3-step PCR cycle, following a short primer anneal step (usually done at 60°C), a higher temperature (such as 72°C) is used for the polymerase extend step. At this higher temperature, primer annealing and activation do not occur, making 3-step PCR less efficient. If 3-step PCR is desired, increasing the length of primer annealing step or increasing the RNase H2 concentration can compensate. Alternatively, 2-step PCR can
    be performed at higher temperature, which will require making the primers longer to increase their Tm to the desired reaction temperature (such as 72°C).
  2. Higher amounts of RNase H2 may be needed for reactions performed at temperatures below 55°C. P.a. RNase H2 has highest activity in the range of
    60–70°C.
  3. Using higher amounts of RNase H2 allows annealing/extension times to be decreased, while decreasing the amount of RNase H2 requires extending the duration of annealing/extension times.

RNase H2 Enzyme Product Information

Certificates of Analysis

Material Safety Data Sheets (MSDSs)