Application guide for rhpcr

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Application guide for rhpcr rhpcr Primers Description Terminal bases* Synthesis scale Purification rhprimer GEN1 rddddmx 100 nmol rhprimer GEN2 rdxxdm 100 nmol Standard desalting RNase H2 Enzyme and

1 Application guide for rhpcr rhpcr Primers Description Terminal bases* Synthesis scale Purification rhprimer GEN1 rddddmx 100 nmol rhprimer GEN2 rdxxdm 100 nmol Standard desalting RNase H2 Enzyme and Buffer Descriptions Size Catalog # RNase H2 Enzyme Kit 50 U RNase H2 (2 U/µL) 2 ml Dilution Buffer Contents RNase H2 Enzyme, Small 50 U at 2 U/µL RNase H2 Enzyme, Large 500 U at 20 U/µL RNase H2 Enzyme Dilution Buffer 2 ml RNase H2 Enzyme Dilution Buffer 10 x 2 ml * r = RNA base (match to target); D = DNA base (match to target); M = DNA base (mismatch to target); x = C3 spacer. Standard desalting is sufficient for most situations. For experiments with long primers or requirements for above average purity, RNase-free HPLC purification is available for an additional fee. 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. Introduction: rhpcr RNase H2 enzyme PCR cycling conditions Master mixes Alternative: polymerases and reaction mixes Reference Revision history rhpcr IDT scientists developed RNase H2 dependent PCR (rhpcr) to increase PCR specificity and eliminate primer dimers by using RNase H2 from Pyrococcus abyssi (P.a.) and blocked primers that contain a single ribonucleotide residue (Figure 1). After the primer hybridizes to the target DNA, the RNase H2 enzyme activates the blocked primers by cleaving the primer at the 5 end of the RNA base. Because the primers must hybridize to the target sequence before they are cleaved, they are unable to form primer dimers. Also, the required high target complementarity reduces amplification of closely related sequences. Superior allele discrimination is achieved when the mismatched base is positioned opposite the RNA base [1]. Figure 1. RNase H-Dependent PCR (rhpcr).

P.a. is an extreme thermophile, so the P.a. RNase H2 enzyme has the highest activity between 60 C and 70 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 than at optimal temperature conditions).

Thus. P.a. RNase H2, with its unique ability to be used in PCR at high temperatures, confers "hot start" character to the PCR.

2 P.a. is an extreme thermophile, so the P.a. RNase H2 enzyme has the highest activity between 60 C and 70 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 than at optimal temperature conditions). Cleavage of the primer will only occur if the rhprimer is bound to its perfectly complementary target, and only cleaved primers are extendable by the polymerase present in typical PCRs. P.a. RNase H2 is functional in most PCR buffers and can be added directly to the PCR master mix. Thus. P.a. RNase H2, with its unique ability to be used in PCR at high temperatures, confers "hot start" character to the PCR. The enzyme functions in real time, making the method a seamless transition from standard PCR with primer cleavage occurring in the background during each annealing/extension cycle [1]. To summarize, the procedure for rhpcr is similar to standard qpcr, but requires blocked-cleavable primers (rhprimers) (Figure 2) and the addition of RNase H2 enzyme to the master mix. This application guide describes how to optimize rhpcr. For information about rhprimer design considerations, visit GEN1 GEN2 Figure 2. Overview of rhprimer GEN 1 and GEN2 Designs. D = DNA bases that match target; M = DNA base that is a mismatch to target; r = RNA base that matches target; x = C3 spacer. RNase H2 enzyme P.a. RNase H2 is available at 2 concentrations, 20 U/µL and 2 U/µL. Before use, dilute the enzyme with RNase H2 Enzyme Dilution Buffer. Using insufficient enzyme will lower reaction efficiency and will require additional PCR cycles. Excess enzyme will decrease specificity, removing the benefit of performing rhpcr. Table 1 provides guidelines for determining how much RNase H2 to use in your experiments. Application guide for rhpcr 2

3 Experimental reagent or condition Recommendations rhprimer GEN1 Use 2 10 mu RNase H2 per 10 µl reaction For larger reactions, proportionally scale up the amount of enzyme used (e.g., use 5 25 mu RNase H2 per 25 µl reaction) Titration experiments: Start with 5 mu per 10 µl reaction and titrate the enzyme up or down so that reaction efficiency is similar to control reactions using unmodified primers. rhprimer GEN2 Use mu RNase H2 per 10 µl reaction For larger reactions, proportionally scale up the amount of enzyme used (e.g., use mu RNase H2 per 25 µl reaction) Titration experiments: Start with 100 mu per 10 µl reaction and titrate the enzyme up or down so that reaction efficiency is similar to control reactions using unmodified primers. RNA nucleotide in blockedcleavable rhprimer If one of the primers, typically an rhprimer GEN2, contains an ru residue at the cleavage site, it might be necessary to use more RNase H2 than for rhprimers that contain ra, rg, or rc. Multiplex rhpcr Use more RNase H2 for multiplex reactions than for singleplex reactions. Recommended: optimize multiplex reactions using enzyme titration experiments. The amount of RNase H2 needed for a multiplex reaction varies with the number of amplicons being detected, primer concentration, and buffer composition. Annealing/extension temperature Annealing/extension time 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 C. Using higher amounts of RNase H2 allows annealing/extension times to be shortened, while using lower amounts of RNase H2 requires increased annealing/extension times. Table 1. Recommendations for optimizing RNase H2 concentration. PCR cycling conditions We recommend using 2-step PCR protocols (Table 2). During PCR, the blocked-cleavable primers anneal to the target and are activated (by RNase H2 cleavage) during the annealing phase of the reaction. In 2-step PCR, the annealing phase also serves as the polymerase extension phase, so this phase is longer, providing for the highest amount of primer activation. In 3-step PCR, following a short primer annealing step (usually done at 60 C), a higher temperature (such as 72 C) is used for the polymerase extension step. At this higher temperature, primer annealing and activation do not occur, making 3-step PCR less efficient. Hot-start polymerase activation Denaturation Annealing, primer cleavage, extension Step Cycles Temperature ( C) Time min (3 min) (45) sec sec (30 sec) Hold 1 4 Up to 24 hr Table 2. Example of cycling conditions. Where there are recommended ranges, IDT standard cycling conditions are specified in parentheses. 3

4 Master mixes IDT scientists have tested compatibility of P.a. RNase H2 with many commercial 5 nuclease PCR master mixes using manufacturers' recommended cycling conditions (Tables 3). Master mixes containing SYBR Green and other intercalating dyes have also been tested (Table 4). Most master mixes and polymerases perform well; however, empirical testing of each mix or polymerase is required because buffer composition affects reaction performance (see Table 1). Master mix Applied Biosystems TaqMan Gene Expression Master Mix Amount of RNase H2 per 10 µl reaction (mu) rhprimers GEN1 (rddddmx) rhprimers GEN2 (rdxxdm) Bio-Rad iq Multiplex Powermix Quanta PerfeCTa Multiplex qpcr SuperMix Table 3. Compatibility of rhprimers With Selected Commercial Master Mixes for 5 Nuclease Assays. All assays were run using the manufacturer s cycling conditions. We have tested additional master mixes. Contact ApplicationSupport@idtdna.com for more information about master mixes not included in this table. Master mix Amount of RNase H2 per 10 µl reaction (mu) rhprimers GEN1 (rddddmx) rhprimers GEN2 (rdxxdm) Agilent Brilliant II SYBR Green Master Mix Bio-Rad iq SYBR Green SuperMix Promega GoTaq qpcr Master Mix Table 4. Compatibility of rhprimers with intercalating dye based master mixes. All assays were run using the manufacturer s cycling conditions. We have tested additional master mixes. Contact ApplicationSupport@idtdna.com for more information about master mixes not included in this table. Alternative: polymerases and reaction mixes This section provides information about setting up rhpcrs using individual polymerases and reaction components. Polymerases We have tested the compatibility of a variety of commercial DNA polymerases with rhpcr. With the exception of the polymerases listed in Table 5, most polymerases performed well. However, empirical testing of each polymerase is required because buffer composition affects reaction performance (see Table 1). GEN1: In general, high-fidelity polymerases with 3 -exonuclease activity perform poorly with rhprimers GEN1 (rddddmx). The exonuclease function of these polymerases removes the 3 blocking group from the rhprimer, which allows amplification to occur in the absence of RNase H2 and effectively eliminates the benefits of using blocked, cleavable primers. GEN2: The 3 block included in rhprimers GEN2 (rdxxdm) is refractory to degradation and provides primer compatibility with high fidelity 3 -exo polymerases. Application guide for rhpcr 4

5 rhprimers GEN1 rhprimers GEN2 DNA polymerase * Notes DNA polymerase * Notes Agilent PfuUltra High-Fidelity No amplification KAPA Biosystems KAPA HiFi Hotstart Not recommended NEB 9 Nm Not recommended NEB Q5 Hot Start High-Fidelity Not recommended NEB Deep VentR Blocked primers are cleaved without RNase H2 Syzygy (Empirical Bioscience) SyFi Not recommended NEB Tli Inconsistent results NEB VentR Blocked primers are cleaved without RNase H2 Thermo Scientific Phire Hot Start >10 mu RNase H2 required Thermo Scientific Phusion High- Fidelity Blocked primers are cleaved without RNase H2 * IDT standard cycling conditions [3 min, 95 C; 45 x (10 sec, 95 C; 30 sec, 60 C)] and the buffers recommended for each polymerase were used. Table 5. DNA polymerases that are not compatible with rhpcr. Reaction mixes Ensure that a minimum, final concentration of 0.01% Triton X100 (or equivalent non-ionic detergent) is 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 when detergent drops below this level. Table 6 provides a starting point for a homemade rhpcr mix, when using rhprimer GEN1 designs. Component Final concentration Table 6. Example of a homemade rhpcr mix for rhprimer Tris ph mm GEN1. See Tables 3 4 for more information about reactions using commercially available master mixes, when using KCl 50 mm rhprimers GEN 1 or GEN2. MgCl 2 dntps 3.0 mm 0.8 mm (0.2 mm each) Triton X % Forward primer Reverse primer Target DNA Taq polymerase P.a. RNase H2 200 nm 200 nm (variable) 0.5 U 0.5 mu/µl (1 µl of a 5 mu/µl stock) Final volume 10 µl 5

6 Reference 1. Dobosy JR, Rose SD, et al. (2011) RNase H-dependent PCR (rhpcr): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers. BMC Biotechnol, 11:80. Revision history Version Date Description of changes 4 March 2017 Updated Table 4 with current product name 3 June 2015 Simplified master mix recommendations for the most current primer designs (GEN1: rddddmx; GEN2: rdxxdm) Deleted information related to IDT cycling conditions for commercially available master mixes Simplified DNA polymerase recommendations for use with homemade master mixes. Updated title to match new document templates Updated IDT address for customer support 2 January 2015 Moved content to a PDF format Reorganized content Added short introduction 1 July 2013 Original information posted online (html) For additional assistance with your rhpcr experiments, contact ApplicationSupport@idtdna.com. For Research Use Only. Not for use in diagnostic procedures Integrated DNA Technologies, Inc. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see PCR AG, 03/17 Integrated DNA Technologies, Inc 1710 Commercial Park Coralville, Iowa Application guide for rhpcr 6

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