RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers
© Dobosy et al; licensee BioMed Central Ltd. 2011
Received: 27 April 2011
Accepted: 10 August 2011
Published: 10 August 2011
The polymerase chain reaction (PCR) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences.
Pyrococcus abyssi (P.a.) RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR). Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs), rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair.
rhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection.
Quantitative PCR (qPCR) is usually performed in real-time mode using fluorescence detection methods. In one commonly used format (the 5'-nuclease assay), qPCR involves three oligonucleotides wherein the forward and reverse primers direct DNA amplification spanning the hybridization site for a third fluorescently labeled oligonucleotide probe. The probe typically contains a fluorescence reporter dye and a quencher. Separation of the reporter and quencher due to cleavage of the probe by the 5'-nuclease activity of the DNA polymerase leads to an increase of fluorescence and a detectable signal [1–3]. Quantitative PCR can also use nucleic acid binding dyes such as SYBR® Green or Eva Green® that increase fluorescence in the presence of double-stranded DNA (dsDNA). Nucleic acid binding dye systems use only two oligonucleotides, the forward and reverse primers, which direct amplification of the target. Once amplification has occurred, the dye binds to the double stranded DNA and generates a fluorescent signal without the need for a third dye-labeled oligonucleotide probe. Dye binding assays are less expensive and are very convenient; however, they are inherently less specific than three-oligonucleotide systems since signal is generated from any amplification event.
Formation of primer-dimers and off-target amplification are common problems in PCR [4–6]. These competing side reactions are a particular problem with low copy number targets due to the high number of cycles required for amplification and in multiplex assays where many different primers must function well together. While "primer-dimers" are often thought to arise from self-amplification of primers due to overlapping 3'-ends, these species can also be generated when there is little apparent complementarity between the primers . More complex oligomeric products of greater length than primer-dimers are also observed in some cases . The formation of primer-dimers can give rise to a false positive signal in dye-binding qPCR assays, and can lead to false negative results by consumption of primers and other reaction components. Several methods can be used to reduce these undesired side reactions, or mitigate their effects. Physical barrier methods can be used to separate reaction components until an elevated temperature is reached [9, 10]. Use of a chemically or antibody inactivated "hot-start" polymerase can alleviate mis-priming at low temperature, but at significant additional cost [11–14]. "Nesting" of primers can detect the desired product among the previously amplified PCR products, but this technique is not applicable to qPCR. Melt-curve analysis done as an additional end-point step in dye-binding qPCR assays can help demonstrate assay specificity by revealing the existence of multiple amplicons, but cannot prevent or limit their formation. Often several assays must be designed and empirically tested before one is found that does not result in multiple melt peaks.
A wide variety of approaches have been employed to confer single-base specificity to PCR assays with the goal of detecting single nucleotide polymorphisms (SNPs) [15, 16]. Assays have been based on either of two methods: differential amplification of the variant alleles (allele-specific PCR, or ASPCR) or discrimination between the alleles following or concurrent with unbiased amplification of the target sequence. The most common format for detection concurrent with unbiased amplification is the 5'-nuclease assay [1, 17]. In that case, a fluorescence-quenched probe, which is degraded by the 5'-nuclease activity of the DNA polymerase, is designed to bind preferentially to the match sequence relative to a mismatch sequence. In order to distinguish effectively between hybridization of an exact match and a single base pair mismatch, relatively short probes, 12-16 bases in length, are needed. To achieve binding of the probe under conditions of the extension reaction with temperatures typically between 55°C and 70°C, modified bases such as locked nucleic acids (LNAs) or pendant groups such as a minor groove binder (MGB) are incorporated into the oligonucleotide to increase the T m [18–20].
In ASPCR, the SNP is positioned at or near the 3'-end of the primer such that a mismatch with the template inhibits initiation of DNA synthesis. Even with careful optimization of reaction conditions, the success rate is highly variable. Assays can be improved by incorporating modified bases or by introducing a secondary mismatch within the primer [21–24]. The most serious shortcoming of this assay format is that once extension has occurred off of a mismatched target, the primer becomes incorporated in the amplicon. After the newly synthesized strand is copied, the primer forms a perfect match with the template and no further discrimination can be achieved. Even if the efficiency of replication of the template is reduced 100-fold due to the mismatch, there would only be a differential amplification of 6-7 cycles between alleles.
Oligonucleotides were synthesized at Integrated DNA Technologies (Coralville, IA) using standard phosphoramidite chemistry. All sequences are listed in Table S1 (Additional file 1).
Cloning and purification of P.a. RNase H2
Cloning, expression, and purification of recombinant Pyrococcus abyssi RNase H2 are fully described in the additional methods (Additional file 2). Briefly, the sequence was obtained from public databases  and the open reading frame was codon optimized for expression in E. coli. A synthetic gene was made and cloned into a shuttle plasmid by the Synthetic Biology group at Integrated DNA Technologies. The gene was transferred to the pET-27b(+) expression vector (Novagen, Madison, WI), which introduced a His-Tag® into the open reading frame. The recombinant protein was expressed in the E. coli strain BL21 (DE3). Bacterial lysates were purified using nickel affinity chromatography. Stock solutions of the enzyme were stored in Buffer A (10 mM Tris pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.1% Triton X-100, and 50% glycerol) at -20°C. The enzyme has been stored under these conditions for over 2 years without detectable loss of activity.
RNase H2 cleavage assays
RNase H2 activity was measured with four duplex substrates, one for each RNA base, having a 14-1-15 design with 14 DNA bases, 1 RNA base, and 15 DNA bases on one strand with complementary DNA bases on the opposite strand. The sequences are shown in Table S1 in the Additional file 1. RNase H2 cleavage reactions were performed with 40 pmoles substrate in Mg Cleavage Buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 4 mM MgCl2, 10 μg/mL BSA, 0.01% Triton X-100) for 20 minutes at 70°C. Reactions were stopped with the addition of EDTA to a final concentration of 10 mM. Reaction products were resolved on a 15% polyacrylamide/7 M urea denaturing gel and visualized on a UV transilluminator after staining for 30 min with 1× GelStar™ Nucleic acid stain (Lonza, Basel, Switzerland).
The effect of single base pair mismatches on substrate cleavage by P.a. RNase H2 was studied using 34 duplex substrates. The perfect match substrate (S-rC 14-1-15) was a 30 mer with the sequence 5'-CTCGTGAGGTGATGcAGGAGATGGGAGGCG-3', having all complementary DNA bases on the opposite strand. DNA bases are shown in uppercase and the one RNA base is shown in lowercase. The region studied in the mismatch analysis is underlined. The ribo-C containing strand was paired with 33 different mismatch complements where every possible single base mutation was introduced at each of the 11 positions indicated above (positions -5 to +5 relative to the RNA base). Cleavage reactions consisted of 2 pmoles of unlabeled substrate spiked with 40 fmoles of 5'-32P-labeled substrate and 0.3 mU of P.a. RNase H2. (1 Unit of P.a. RNase H2 is the amount of enzyme needed to cleave 1 nmole of S-rC 14-1-15 per minute at 70°C in Mg Cleavage Buffer, and corresponds to approximately 5 fmoles of enzyme.) Reactions were incubated for 20 minutes at 70°C in 20 μL of Mg Cleavage Buffer. Reaction products were separated by PAGE on a 15% polyacrylamide/7 M urea denaturing gel and visualized using a Packard Cyclone™ Storage Phosphor scanner (Packard Biosciences, Meriden, CT). The percentage of cleaved vs. uncleaved substrate was determined using the OptiQuant™ ver. 4.00 software (Packard Biosciences, Meriden, Connecticut).
Pyrococcus abyssiRNase H2 heat stability assays
RNase H2 temperature stability was assessed in triplicate by pre-incubating aliquots of P.a. RNase H2 at 95°C for increasing periods of time, ranging from 0 to 90 minutes. Thermal inactivation of the RNase H2 enzymatic activity was detected by reduced substrate cleavage efficiency. Cleavage reactions consisted of 2 pmoles of unlabeled S-rC 14-1-15 spiked with 40 fmoles of 5'-32P-labeled substrate and 0.1 mU of the pre-incubated P.a. RNase H2 enzyme. Detection and quantification of reaction products were performed as outlined above.
Pyrococcus abyssiRNase H2 temperature dependence studies
The enzymatic activity of P.a. RNase H2 was assessed at different reaction temperatures ranging from 30°C to 70°C. Cleavage reactions consisted of 2 pmoles of unlabeled S-rC 14-1-15 spiked with 40 fmoles of 5'-32P-labeled substrate and 0.25 mU of P.a. RNase H2 in 20 μL of Mg Cleavage Buffer. Reactions were allowed to proceed for 20 minutes and products were separated by PAGE on a 15% polyacrylamide/7 M urea denaturing gel and quantified as described above.
Optimization of rhPCR primer design and amplification protocols using a synthetic amplicon
Optimization of blocked-cleavable primer design, enzyme concentration, buffer composition, and cycling parameters was done using a synthetic template. Reaction products were examined by separation on a 15% polyacrylamide/7 M urea denaturing gel, stained for 30 min with 1× GelStar™ Nucleic acid stain (Lonza, Basel, Switzerland), and visualized under UV excitation. In addition, the template was amplified in qPCR assays using either SYBR® Green or a dual-labeled fluorescence-quenched hydrolysis probe for detection. The target oligonucleotide that defines this amplicon is a 103mer synthetic sequence that is not homologous to any known gene (see Table S1). Reactions were run on a Roche Lightcycler® 480 in 384 well plates using 10 μL reaction volumes. Cycling conditions included an initial 5 minute soak at 95°C, followed by 45 cycles of 10 seconds at 95°C and 30 seconds at 60°C. For some experiments, the dwell time at 60°C was varied between 30, 60, and 120 seconds. Reactions were minimally performed in triplicate.
SYBR® Green reactions (10 μL) consisted of 5 μL of 2× BIO-RAD iQ™ SYBR® Green Supermix (BIO-RAD, Hercules, CA), 200 nM each of the forward and reverse primers, 20 to 2 × 106 copies of the synthetic oligonucleotide template, with varying amounts of P.a. RNase H2. The iQ™ SYBR® Green Supermix contains 3 mM MgCl2. 5'-nuclease probe hydrolysis reactions consisted of 0.4 U iTaq DNA polymerase (BIO-RAD, Hercules, CA), 1 μL 10X iTaq™ buffer (BIO-RAD, Hercules, CA), 3 mM MgCl2, 800 μM dNTPs, and 200 nM each of the forward and reverse primers and the probe, 20 to 2 × 106 copies of the synthetic oligonucleotide template, with varying amounts of P.a. RNase H2. The quantification cycle number (Cq) was determined using the absolute quantification/2nd derivative method .
Hepatitis C Virus (HCV) Primer-Dimer Studies
Primer-dimer studies employed a HCV amplicon that was previously shown to produce significant primer-dimer artifacts . A 242 base pair HCV target was made as a synthetic gene and cloned in a plasmid by the Synthetic Biology group at Integrated DNA Technologies (Coralville, IA). qPCR assays were performed in triplicate on a Roche Lightcycler® 480 in 10 μL reaction volumes with 5 μL of 2× DyNAmo™ SYBR® Green qPCR kit (NEB, Ipswich, MA), 200 nM of each primer, 2 × 104 copies of the synthetic HCV template plasmid with or without 2 ng rat spinal cord cDNA, and 0 or 2.6 mU of P.a. RNase H2. The DyNAmo™ SYBR® Green qPCR reaction mix contains 2.5 mM MgCl2 final concentration. Cycling conditions consisted of an initial 5 minute soak at 95°C, followed by 50 cycles of 95°C for 30 seconds and 60°C for 30 seconds. Primer sequences and the sequence of the HCV amplicon are shown in Table S1. Products were separated by PAGE on a 15% non-denaturing polyacrylamide gel, stained for 30 min with 1× GelStar™ Nucleic acid stain (Lonza, Basel, Switzerland), and visualized under UV excitation.
Studies of the specificity of rhPCR using mammalian cDNA
The performance and specificity of rhPCR with complex nucleic acid samples was first examined using a qPCR assay designed against a human gene target (HRAS). Amplification reactions were compared using human cDNA (prepared from HeLa cell total RNA) and rat cDNA (prepared from rat spinal cord total RNA). Blocked-cleavable and control primers that produce a 340 bp amplicon from the human HRAS gene (NM_005343) were synthesized (see Table S1). HRAS qPCR assays were performed on a Roche Lightcycler® 480 in 10 μL reaction volumes, containing 1 μL P.a. RNase H2 (1.3 mU), 5 μL 2× BIO-RAD iQ™ SYBR® Green Supermix (BIO-RAD, Hercules, CA), 2 ng of rat spinal cord cDNA or 2 ng HeLa cDNA, and 200 nM of each primer. Cycle conditions included an initial 5 minute soak at 95°C, followed by 60 or 90 cycles of 10 seconds at 95°C and 90 seconds at 60°C. All reactions were run in triplicate.
Analysis of the SMAD7 rs4939827 SNP with rhPCR
Reactions were performed on a Roche Lightcycler® 480 in 10 μL final volume with 5 μL 2× BIO-RAD iQ™ SYBR® Green Supermix, 200 nM forward and reverse primers and 2 or 20 ng of genomic DNA (GM18562 or GM18537, obtained from the Coriell Institute for Medical Research Cell Repository (http://ccr.coriell.org/)). Reactions were performed in triplicate. One μL (2.6 mU) of P.a. RNase H2 in Buffer A or Buffer A without RNase H2 was added to each reaction. Thermal cycling was performed using an initial 5 minute soak at 95°C followed by 45 cycles of 10 seconds at 95°C and 30 seconds at 60°C. Additional experiments were performed varying the cycling parameters and concentration of P.a. RNase H2 as outlined in the Results section. Certain studies were performed using up to 100 cycles. All reactions were performed using the same unblocked reverse primer. Forward primers included unmodified allele-specific primers as well as blocked-cleavable primers of different designs. A non-discriminatory unmodified primer served as a control. Cq and ΔCq values were computed as described above.
Recombinant RNase H2 from Pyrococcus abyssi
The rnhb gene coding for the Type II RNase H from Pyrococcus abyssi has been identified previously [27, 28]. We produced and purified recombinant Pyrococcus abyssi RNase H2 from E. coli as outlined in the additional methods (Additional file 2). As expected, the enzyme was found to cleave heteroduplex substrates having a single ribonucleotide comprising any of the four RNA bases. Mass spectrometry analysis confirmed that cleavage occurred on the 5'-side of the RNA residue, yielding one fragment with a free 3'-OH group and a second with a 5'-ribonucleotide phosphate (see Figure S1, Additional file 3). Importantly, single-stranded RNA-containing oligonucleotides were not cleaved or otherwise degraded, demonstrating the absence of any contaminating nuclease activity in the enzyme preparation.
Magnesium requirements were optimized for P.a. RNase H2 by examining the dependence of the cleavage rates on Mg2+ concentration for the single rC containing 30mer heteroduplex substrate S-rC 14-1-15 at 70°C. Maximum activity was achieved around 4 mM Mg2+ and high levels of activity were seen in the range of 1 to 10 mM Mg2+, similar to other RNase H2 enzymes characterized [28–30]. The enzyme maintained over 90% activity at 2 mM Mg2+, and only dropped to 61% of optimal activity in 1 mM Mg2+ (see Figure S2, Additional file 4). No cleavage was observed in the absence of divalent cations. As with other Type II RNase H enzymes [29, 30], P.a. RNase H2 also has the ability to utilize Mn2+ and Co2+ in place of Mg2+ (data not shown).
The 5'-reaction product formed upon cleavage with RNase H2 has the structure of a normal primer used to initiate PCR reactions: the sequence is entirely DNA and ends in a 3'-hydroxyl group. Reaction conditions for P.a. RNase H2 are compatible with the buffers commonly employed in PCR (Mg2+ concentrations, pH, etc.). If the enzyme has sufficient thermal stability and a high enough turnover rate, then it should be possible to perform primer cleavage/activation in real time during PCR.
Thermal stability and temperature dependence of Pyrococcus abyssiRNase H2
The temperature-dependence of the activity of P.a. RNase H2 was studied by comparing the ability of the enzyme to cleave the substrate S-rC 14-1-15 at different temperatures between 30°C and 70°C (Figures 2B and 2C). Minimal cleavage (< 1%) of the substrate was observed at 30°C, indicating that P.a. RNase H2 is effectively inactive at room temperature. In contrast, the P.a. RNase H2 enzyme cleaved over 70% of the total substrate at 70°C. The enzyme retained 64% relative activity at 60°C and 23% relative activity at 50°C. Below 50°C, the enzyme rapidly lost activity. These results demonstrate that P.a. RNase H2 is highly active throughout the range of temperatures commonly used in PCR and is only minimally active at ambient temperatures.
Effect of single base pair mismatches on substrate cleavage by P.a. RNase H2
rhPCR assays using P.a. RNase H2
A 103 base synthetic oligonucleotide template was used as an artificial amplicon for optimization of primer design for rhPCR. Initial testing of P.a. RNase H2 using radiolabeled substrates indicated that 8 to 10 base pairs of duplex DNA 5'-to the RNA residue and 4 to 5 base pairs of DNA duplex 3'-to the RNA base were necessary for optimal cleavage (data not shown). In rhPCR (Figure 1), the 5'-cleavage product must be a functional primer. This ensures that more than 10 bases of DNA are always present on the 5'-side of the RNA residue. The synthetic amplicon system was employed to test the structural requirements on the 3'-side of the RNA residue for efficient cleavage by RNase H2 during thermocycling.
A single unmodified forward (For) PCR primer was paired with a set of blocked-cleavable reverse (Rev) primers having different numbers of DNA residues 3'-to the RNA base. The Rev primers all contained the same 22 base primer domain on the 5'-end followed by a single ribonucleotide (rU) plus 2, 3, 4, 5, or 6 DNA bases complementary to the target and ended in a 3'-terminal dideoxy-cytosine (ddC). The ddC residue blocks primer extension; primer function is activated by RNase H2 cleavage 5'-to the ribonucleotide residue. After 45 cycles (10 seconds at 95°C followed by 30 seconds at 60°C), reaction products were separated by PAGE. No PCR products were produced using blocked primers in the absence of RNase H2, indicating that the Rev primers were inactive and did not spontaneously cleave to give rise to an active species under PCR conditions (see Figure S3, Additional file 5). In the presence of RNase H2, amplification products were not seen for the 2-DNA or 3-DNA primers whereas the correct amplification product was observed using the 4-DNA, 5-DNA, and 6-DNA primers, consistent with earlier studies of substrate cleavage by the enzyme under steady state conditions. Identical results were observed when the 3'-terminus was blocked with a C3 propanediol spacer instead of ddC.
Elimination of primer-dimer formation using rhPCR
Primer-dimer and other misamplification events are a common problem in PCR and are a particular issue in SYBR® Green qPCR assays where false-positive signal detected from misamplified products can occur. Sequence analysis, careful design of PCR primers, and use of hot start methods can reduce the frequency of primer-dimer and misamplification events, but by necessity some assays must be designed in the contexts of sequences that are not ideal. This problem is further magnified in the setting of multiplex PCR where several different primer pairs must function well together. Due to the low level of activity of P.a. RNase H2 at room temperature (Figures 2B and 2C), use of this enzyme in rhPCR should result in a primer-based hot-start reaction. Moreover, amplification requires that the primers anneal and form a suitable heteroduplex substrate for RNase H2 cleavage, which should impart greater specificity to the start of the reaction than is provided by hybridization of the primer to the target alone. Taken together, these two factors should mitigate the formation of primer-dimers.
Improved specificity of rhPCR with complex nucleic acid samples
The human HRAS control and blocked-cleavable primers are aligned against the corresponding rat Hras sequences in Figure 6B. In spite of multiple mismatches present in both the forward and reverse control primers, amplification from rat cDNA was observed with only an 11 cycle delay compared to the perfect match. As a result of the presence of mismatches around the site of cleavage (the ribonucleotide base), both primers form poor substrates for RNase H2, conferring an additional level of specificity to rhPCR and giving greatly improved performance compared to the control primers.
Detection of single base pair mismatches with rhPCR
The experiments shown in Figure 7 examined the effects of a mismatch at the RNA base (position "0" as defined in Figure 3). The effects of mismatches at the "-1" and "+1" positions on the rhPCR assay were also studied. For these experiments, blocked-cleavable Rev primers were synthesized where the RNA base was held constant and the DNA base at position "-1" or "+1" was varied. Primers were made for all 4 RNA bases with all 4 DNA bases at these two flanking positions. Complementary oligonucleotide targets were synthesized as before. SYBR® Green qPCR assays were performed and ΔCq values measured between the perfect match and every possible single base mismatch. Results for the 64 primer/target pairs with a mismatch at position "-1" are shown in Figure S4 (Additional file 6) and those with a mismatch at position "+1" are shown in Figure S5 (Additional file 7). Standard errors of the ΔCq values were less than or equal to 0.3 cycles in all cases. Mismatch discrimination varied from 0.8 to 16.1 cycles for the "-1" series, with an average ΔCq value of 7.7. For the "+1" series, ΔCq varied from 0.2 to 13.8 cycles, with an average value of 6.6. Although mismatch discrimination varied with sequence context, overall the greatest specificity was achieved by placing the mismatch opposite to the RNA base (position "0").
Application of rhPCR to detect single nucleotide polymorphisms in human genomic DNA
Mismatch discrimination for various primer designs at the SMAD7 rs4939827 SNP locus
Studies using the synthetic amplicon system suggested that placing the SNP site opposite the RNA base (position "0") generally gave the highest mismatch discrimination; however, when the SNP site was placed at positions "-1" or "+1" the ΔCq was also very favorable in some cases. To determine the optimal position of the SNP site within the blocked-cleavable primer in the sequence context of the SMAD7 rs4939827 SNP, primers were synthesized for both the C and T alleles placing the SNP site at the "-1" and "+1" positions relative to the RNA base. As with the synthetic template, placing the SNP at the RNA base (position "0") gave the best results. Blocked-cleavable primers with the SNP at the "-1" position performed very poorly with ΔCq values of less than 2 (Table 2). These results were not statistically different from results obtained with the unmodified primers. Placing the SNP site at the "+1" position gave intermediate results with ΔCq values of 8.8 and 2.6 for the "C-allele" and "T-allele" primers, respectively. These results represented a statistically significant improvement over the unmodified primers but reflected decreased specificity when compared with blocked-cleavable primers having the mismatch placed opposite the RNA residue (P < 0.05 for both alleles).
In all of these experiments, thermocycling was performed using a 60°C anneal/extension temperature. Standard unmodified allele-specific primers may be more dependent upon using an optimized reaction temperature than the rhPCR method, so the experiments were repeated with anneal/extension temperatures ranging from 60°C to 70°C. Results with the unmodified primers were slightly better at elevated anneal temperatures, but in no case did the ΔCq between the matched and mismatched targets exceed 4 cycles (data not shown). Experiments performed using the blocked-cleavable primers also showed little improvement in mismatch discrimination by increasing the reaction temperature above 60°C.
In traditional qPCR and PCR reactions, it is occasionally necessary to employ a primer annealing temperature lower than 60°C due to sequence context constraints imposed by the natural nucleic acid targets. To determine whether the lower enzymatic activity of P.a. RNase H2 seen at temperatures below 60°C would affect the efficiency of rhPCR or mismatch discrimination, amplification reactions were repeated with the SMAD7 rs4939827-specific blocked-cleavable primers at annealing temperatures of 55°C or 50°C (see Table S2 in Additional file 8). Only a small decrease in amplification efficiency of rhPCR was observed at 50°C with no significant effect on mismatch discrimination. Thus, in spite of the reduction of activity of the P.a. RNase H2 enzyme as the reaction temperature is lowered from 60°C to 50°C (see Figure 2C), the efficiency and specificity of rhPCR remains remarkably stable throughout this temperature range.
Use of rhPCR in genotyping
The rhPCR assay was clearly effective at discriminating these 3 genotypes when run with the benefit of the high precision offered by real-time detection. For high throughput applications, it is more efficient to process plates in batch format and read fluorescence in an end-point mode. The above experiment was therefore repeated running the reaction for 35 cycles and fluorescence was measured at this point; raw fluorescence values were plotted for each sample and are shown in Figure 8B. Each DNA sample was run in triplicate, so the fluorescence measurements include 93 data points. Once again, all 31 human DNA samples (93 PCR reactions) were easily stratified into 3 classes (homozygote C/C, homozygote T/T, and heterozygote C/T) and each genotype was correctly identified. As more amplification cycles are performed, signal from mismatched targets will eventually increase. At 40 cycles the correct genotypes of all samples were still easily identified, but by 45 cycles the boundaries between homozygote and heterozygote samples began to merge (data not shown). By limiting the reaction to 40 cycles or less (with an input of 2 ng of genomic DNA), rhPCR can be used for genotyping applications in an end-point format.
Primer Cleavage Dependent PCR
Several coupled reaction schemes have been proposed for PCR in which a hybridization dependent primer activation step is linked to primer extension. In the pyrophosphorolysis-activated polymerization (PAP) assay [34, 35], a blocked 3'-terminal nucleotide is cleaved by attack of pyrophosphate (reverse of the polymerization reaction). For this to occur efficiently, high concentrations of pyrophosphate are required which may inhibit some polymerases. The range of blocking groups that can be accommodated at the 3'-terminus is very limited. A 3'-terminal dideoxynucleotide has been utilized in most studies [34, 35]. Of the four bases, only dideoxy-C can be readily incorporated using standard methods of oligonucleotide synthesis, limiting widespread use of this technique.
A coupled PCR assay has been proposed in which a blocked primer is cleaved after hybridization to the target sequence by a nicking restriction endonuclease . A restriction enzyme that has an asymmetric recognition sequence or that cuts only one strand at a hemimethylated site would be required to avoid cleavage of the template. To our knowledge, this reaction scheme has never been demonstrated experimentally. In any event, the requirement that the restriction enzyme recognition sequence be located near the 3'-end of the primer would severely limit the use of this method.
Use of both RNase H1 and RNase H2 to effect primer cleavage in a coupled PCR assay has been reported previously in the patent literature but minimally characterized [37, 38]. Unlike the Type II RNase H enzymes, Type I enzymes will not cleave a substrate having a single RNA residue. At least 3 consecutive RNA residues are required, and 4 for a high level of catalytic activity . Thus, use of a Type I RNase H in rhPCR would require that the primer have at least four consecutive RNA residues. This adds substantially to the cost and complexity of the synthesis of the primer and increases its susceptibility to degradation. The cleaved primer would terminate in two or more RNA residues which can inhibit primer extension and these RNA residues would be incorporated into the amplicon. Sagawa et al.  suggested that the specificity of Type II RNases H would be similar to that of a restriction enzyme and that cleavage, and hence amplification, would be completely prevented if there was a mismatch at the RNA:DNA base pair within the duplex formed between the primer and the template. Although this is not true, as seen in the present study, coupling RNase H2 cleavage to primer extension can be used to greatly boost the specificity of PCR.
Use of P.a. RNase H2 in rhPCR
The thermostability and temperature dependence of P.a. RNase H2 makes it well suited for use in rhPCR. The activity of the enzyme is unaffected by heating at 95°C for 45 minutes (Figure 2A). Critically, this level of resistance to heat inactivation is sufficient for use in PCR, even for reactions requiring an extended number of cycles and a sustained initial incubation at 95°C. To demonstrate this explicitly, P.a. RNase H2 was pre-cycled in PCR buffer for 80 cycles (without primers, dNTPs, or template), then all reaction components were added and rhPCR was performed (data not shown). The reaction efficiency was unchanged, indicating that the enzyme's thermal stability is sufficient to remain active throughout the range of use expected for all PCR applications. A further advantage of P.a. RNase H2 is that it is essentially inactive at temperatures below 30°C (Figure 2B and 2C). This effectively confers a hot start to reactions done using blocked-cleavable primers and any thermostable DNA polymerase. In addition, P.a. RNase H2 has sufficient activity at 50°C to support rhPCR, permitting the reaction to be performed throughout a broad temperature range (Table S2, Additional file 8).
Like other Type II RNase H enzymes, P.a. RNase H2 retains a high level of activity at concentrations of Mg2+ as low as 1 mM [28–30], enabling rhPCR to be performed at all magnesium concentrations typically employed in PCR. The enzyme is able to cleave heteroduplex substrates with a single ribonucleotide comprising any of the four bases. Cleavage also occurs at a riboA:deoxyU base pair (not shown), allowing the uracil-N-glycosylase (UNG) sterilization method to be used in rhPCR assays. Although reaction rates are slightly affected by the identity of the RNA base and the flanking DNA sequence, a single concentration of the enzyme and set of reaction conditions generally can be used regardless of the sequence of the target. The efficiency of primer cleavage is principally determined by the structure of the primer 3'-to the RNA base and the degree of complementarity present near the scissile linkage.
It is important to note that non-specific hydrolysis of the RNA linkage cannot lead to primer activation. Water catalyzed hydrolysis and enzymatic cleavage by contaminating single-strand specific ribonucleases both lead to the formation of a cyclic 2'-3'-phosphodiester at the 3'-terminus. This group blocks the 3'-end of the oligonucleotide and prevents primer extension. Spontaneous hydrolysis of the cyclic phosphodiester gives a mixture of 2'- and 3'-phosphate monoesters; enzymatic hydrolysis yields exclusively the 3'-phosphate. In either case, primer extension remains blocked. Background cleavage of the ribonucleotide linkage is problematic only if it occurs to such an extent that the amount of the primer is substantially depleted. Although divalent cations (including Mg2+) can facilitate water catalyzed hydrolysis of RNA containing oligonucleotides, especially at elevated temperatures , non-enzymatic degradation of primers containing a single RNA residue under thermocycling conditions used in PCR is negligible. If there is significant contamination of a sample with single-stranded ribonucleases, inhibitors such as human placental RNase inhibitor can be included in the reaction mixture as they do not affect the activity of RNase H enzymes. In our experience, this has not been necessary.
Recognition of Substrates having Base-Pair Mismatches by Type II RNase H Enzymes
RNase H2 plays an important role in the removal of RNA residues misincorporated into DNA due either to incomplete removal of RNA primers used to initiate DNA synthesis or polymerase errors [41–45]. Consistent with its role in DNA repair, Type II RNase H enzymes are also able to cleave substrates where there is an RNA:DNA base pair mismatch, but at a rate reduced compared to the corresponding perfect duplex [31, 32, 46–48]. For P.a. RNase H2, the rate of the reaction is decreased by about 10-fold (Figure 3). A decrease in rate of similar magnitude is seen with a mismatch on the 5'-side of the cleavage site (position "-1"). Mismatches at the "-3", "-2", and "+1" positions gave rise to smaller reductions in the cleavage rate. Outside of this region, effects of a base pair mismatch were negligible. In all cases, the only products observed by mass spectrometry, and by electrophoresis using radiolabeled substrates, reflected cleavage on the 5'-side of the RNA residue. More detailed kinetic studies of the effects of mismatches on cleavage rates are in progress.
Enhanced specificity of rhPCR
Coupling cleavage by RNase H2 to primer extension in rhPCR leads to greater specificity both with respect to template independent mispriming events (e.g., primer-dimer formation) and unwanted amplification of related sequences. The formation of primer-dimers is prevented even in assays that are very prone to this side reaction (Figure 5). This feature of rhPCR should be particularly beneficial in multiplex assays. The specificity of the assay with respect to misamplification of homologous sequences is far greater than can be achieved by PCR using unmodified primers. When there are mismatches over or neighboring the RNase H2 cleavage sites of both primers, the ΔCq values observed are extremely large. For the HRAS gene, the ΔCq between the rat and human sequences was greater than 50 cycles. This high degree of specificity should be very useful for the detection of low levels of heterologous DNA in xenogeneic transplant models (e.g., human tumors grown in a mouse host) and in other instances where there are related targets having closely spaced variations in sequence. In SNP detection, where it is necessary to exploit the effect of a single base pair mismatch on cleavage by RNase H2, the assay also shows far greater discrimination than can be achieved with standard allele-specific PCR.
Use of rhPCR for Genotyping
Several groups have employed RNase H in SNP discrimination assays using unbiased amplification of the target sequence linked to cleavage of an RNA-containing probe. Harvey, Han, and colleagues described the use of a thermostable RNase H1 enzyme in genotyping assays where cleavage of a fluorescence-quenched probe having four sequential RNA bases was used to discriminate base identity . The assay was coupled to PCR and cleavage of the probe occurred in real time during thermocycling, as in a 5'-nuclease assay. The Type II RNase H enzymes from Chlamydia pneumonia (C.p.) and from Thermus thermophilus (T.th.) have been used for SNP detection where a Molecular Beacon having a single RNA residue specific for the mutation site was cleaved following PCR in an end-point assay [32, 48, 49].
In rhPCR the discrimination between variant alleles relies on differential amplification of the matched and mismatched target sequences. The specificity of the assay is generally greatest when the RNA residue of the primer is placed over the SNP site. In a model system using "rDDDDx" blocked-cleavable primers, all 12 possible base pair mismatches were readily detected (Figure 7A). The average ΔCq was 10.9 using the same set of reaction conditions in each assay. With unmodified allele-specific primers, the average ΔCq was only 5.4.
The application of rhPCR to genotyping of genomic DNA samples was investigated with the SMAD7 rs4939827 (C/T) SNP locus. Individuals homozygous for the T/T allele are at increased risk for the development of colon cancer. With unmodified allele-specific primers, there was almost no discrimination between the two alleles (Table 2), precluding the use of traditional ASPCR for genotyping at this locus. With rhPCR, the two alleles were easily distinguished. Using "rDDDDx" primers with the RNA residue positioned over the SNP site, the ΔCq between matched and mismatched targets was approximately 12 for both the "T-allele" and "C-allele" specific primers. The three genotypes (C/C, C/T, and T/T) could be distinguished unambiguously. In a blinded study of 31 DNA samples representing different individuals, all were correctly identified. The assay was robust with both real-time and end-point modes of detection (Figure 8).
In studies with both a synthetic template (Figures S4 and S5, Additional files 6 and 7) and the SMAD7 SNP locus (Table 2), rhPCR primers with the mutation site located 5'-to the RNA base (position "-1") were less discriminatory than primers where the RNA base was placed over the SNP site. In the case of the SMAD7 locus, rhPCR primers with the SNP site at the "-1" position provided almost no differentiation between the two alleles, similar to unmodified allele-specific primers. At first, it might seem that placing the mutation site on the 5'-side of the RNA base would be optimal, providing discrimination both at the RNase H2 cleavage step and at the initiation of DNA synthesis. In contrast, with the RNA base opposite the mutation site (position "0"), the primer forms a perfect match to the template after RNase H2 cleavage, offering no opportunity for further distinction between the two alleles. However, in the former case, if primer cleavage and extension do occur on a mismatched template, the alternate allele is incorporated into the extension product and a new amplicon is created which is now a perfect match to the primer. As a result, a 10-fold decrease in the cleavage rate by RNase H2 can contribute at most a 3-4 cycle increase in the Cq value. On the other hand, if the RNA base is positioned opposite the mutation site, the mismatched template should be replicated faithfully. When this occurs, the effect of the mismatch becomes amplified with each cycle and produces a much greater increase in the value of ΔCq. For convenience, Additional file 9 contains a merged set of all of the additional files.
Other applications for thermostable RNase H2 in molecular biology
RNase H2 can also be used to increase the specificity of DNA ligation assays (data not shown). Oligonucleotide ligation assays (OLAs) employ two oligonucleotides (an acceptor oligonucleotide with a reactive 3'-hydroxyl group and a donor oligonucleotide with a 5'-phosphate) which are designed to hybridize adjacent to each other on a complementary target nucleic acid so that DNA ligase can join the two fragments [50–54]. The formation of this new longer species is detectable by a variety of means including PCR and fluorescent bead capture. An added degree of specificity for SNP detection relies upon the ability of DNA ligase to distinguish between a perfect match and mismatched base pair at or near the site of ligation. Donor and/or acceptor oligonucleotides can be designed that are modified to prevent ligation and contain an internal RNA residue near the ligation site. Blocking groups on the acceptor oligonucleotide useful to inhibit ligation are the same as those used to prevent primer extension. As with the blocked-cleavable primers used in rhPCR, cleavage of the blocked-cleavable OLA oligonucleotide will result in a free 3'-hydroxyl which can function as an acceptor for a 5'-phosphate during ligation. Cleavage of the donor oligonucleotide by RNase H2 will result in a 5'-ribophosphate, which will also function efficiently in ligation . This coupled reaction scheme could be employed to improve the specificity of OLAs for nucleic acid detection or genotyping.
In summary, we describe here a PCR method (rhPCR) that couples activation of blocked-cleavable primers by P.a. RNase H2 with amplification. The new rhPCR method simplifies primer design by eliminating the formation of primer-dimers and markedly enhances the specificity of PCR with respect to off-target amplification of closely related sequences. There is no change in workflow and use of a less expensive non-hot start DNA polymerase may actually result in a reduction in assay costs. In addition to its use in genotyping, the assay should find utility in improving the function of highly multiplexed assays where many different primers must work well together without unwanted interactions (primer-dimers, etc.), in rare allele detection where the added specificity of the blocked-cleavable primers enables detection of a desired mutant allele in the face of increasingly large amounts of wild type DNA (manuscript in preparation), and in library construction for Next Generation DNA Sequencing methods to reduce contamination of libraries with primer dimer "blank reads".
This research and open access page charges were supported by funding from Integrated DNA Technologies, Inc.
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