A modified PCR technique coupled with an RNase H enzyme is known as rhPCR. This method uses primers that are modified at 3′ end. A removable amplification block is a modification on the 3’ end. For amplification reaction to be carried the blocked primer should be cleaved. The cleavage is done by the is hyperthermophilic RNase enzyme. The use of RNase H enzyme has added some additional characteristics to the PCR. Firstly, the low activity of the enzymes at low temperature enables a “hot start PCR” without modifications to the DNA polymerase. Secondly, the efficiency of cleavages reduces with mismatches near the RNA residue. The primer-dimer formation is controlled. Alternative splicing variants are detected. High numbers of PCR primers leads to the formation of multiple PCR. SIngle-nucleotide polymorphism is also detectable by this method.
A single RNA residue is modified at or near the 3’-end of the oligonucleotide of the primers. This prevents extension by DNA polymerase. When the hybridization of target DNA sequence and subsequent cleavage by RNase H2 happens the primers get deblocked and activated. The properties of the enzyme of high thermal suitability and turn over makes the reaction possible at a high turnover rate in real time during thermocycling. Cleavage occurs at the 5’-side of the RNA base. It leaves a DNA oligonucleotide with a 3’-hydroxyl that is competent to function as a primer.
Synthetic oligonucleotides are used.
Cloning and characterization of Pyrococcus abyssi RNase H2
Production and purification of P.a. RNase H2
The following protocol is optimized and represents the large-scale preparation.
1) BL21(DE3) bacterial cells were transformed with a pET-27b(+) plasmid containing the E. coli codon-optimized Pyrococcus abyssi RNase H2 gene cloned into the plasmid at the BamHI/HindIII sites.
2) Two x 1 L bacterial cultures were grown to log phase in selective LB media.
3) RNase H2 protein production was induced with1 mM IPTG at 37°C for 6 hours.
4) Cells were harvested at 5,000 rpm for 10 minutes and frozen overnight at -20o C.
5) Cell paste was thawed and 50 mL of Protein Extraction Reagent, 50 kU Lysozyme, and 2500 U RNase-free DNase I was added per liter of original culture.
6) Cell lysate was incubated with rotation at 25°C for 30 minutes. The lysate was centrifuged at 16,000 x g for 30 minutes to pellet insoluble materials, and the soluble supernatant was removed and placed in a fresh tube.
7) DNase I was heat inactivated at 75°C for 15 minutes and insoluble materials were removed by centrifugation at 16,000 x g for 10 minutes.
8) A 4% to 20% SDS-polyacrylamide gel was run with increasing amounts of soluble and insoluble material and Coomassie stained to estimate the quantity of RNase H2 present in both fractions.
9) The heat treatment was found to result in a very effective first step of purification; this material was further purified by capture using a column. Elution was performed with 2 x 6 volumes of elution buffer containing 200 mM imidazole.
10) A 70% ammonium sulfate precipitation was performed to concentrate the purified protein.
11) SDS-PAGE revealed a single band of the expected molecular weight (27.6 kDa) with little contaminating material.
12) The enzyme was dialyzed into Buffer A (10 mM Tris pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.1 % Triton X-100, and 50% glycerol) and stored at -20o C.
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
The activity of RNase H2 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.
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× Nucleic acid stain.
A single base pair mismatches on substrate cleavage by P.a. RNase H2 was studied using 34 duplex substrates. 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’-32Plabeled 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 the 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.
The percentage of the cleaved vs. uncleaved substrate was determined using the Imaging software.
Pyrococcus abyssi RNase H2 heat stability assays
Temperature stability of RNase H2 was assessed in triplicates. The aliquots of P.a. RNase H2 is pre-incubated at 95° C for increasing periods of time, ranging from 0 to 90 minutes. Reduced substrate cleavage efficiency has used a measure of thermal inactivation of the RNase H2 enzymatic activity. 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 abyssi RNase H2 temperature dependence studies
Different reaction temperatures ranging from 30°C to 70°C were used to assess the enzymatic activity of P.a. RNase H2. Cleavage reactions consisted of 2 pmoles of unlabeled S-rC 14-1-15 spiked with 40 fmoles of 5’-32Plabeled 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 Nucleic acid stain, 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 amplicon which is 103mer synthetic sequence is the target oligonucleotide. It is not homologous any known gene. Reactions were run 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 BIO-RAD SYBR Green Supermix, 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 quantification cycle number (Cq) was determined using the absolute quantification/2nd derivative method.
Hepatitis C Virus (HCV) Primer-Dimer Studies
HCV amplicon produces significant primer-dimer artifacts. So this HCV amplicon is used in primer-dimer studies. A 242 base pair HCV target was made as a synthetic gene and cloned into a plasmid. qPCR assays were performed in triplicate in 10 μL reaction volumes with 5 μL of 2× SYBR Green, qPCR kit, 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 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. Products were separated by PAGE on a 15% non-denaturing polyacrylamide gel, stained for 30 min with Nucleic acid stain, and visualized under UV excitation.
Studies of the specificity of rhPCR using mammalian cDNA
To study the specificity of rhPCR a qPCR assay is 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 were synthesized. HRAS qPCR assays were performed on a 10 μL reaction volumes, containing 1 μL P.a. RNase H2 (1.3 mU), 5 μL Green Supermix, 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 10 μL final volume with 5 μL SYBR Green Supermix, 200 nM forward and reverse primers and 2 or 20 ng of genomic DNA (GM18562 or GM18537). 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. 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
Once the enzyme is produced their characteristics are tested to find the purity of the enzyme and their applicability in real time. E. coli is generally used in the production and purification of recombinant Pyrococcus abyssi RNase H2. The enzyme produced can cleave heteroduplex substrates, which has a single ribonucleotide. The nucleotide can be any one of the four RNA bases. The cleavage is found on the 5’-side of the RNA residue by mass spectrophotometric analysis. It yields one fragment with a free 3’-OH group and second with a 5’-ribonucleotide phosphate. Single-stranded RNA was not cleaved. The 5′ reaction product acts as a primer molecule. High levels of enzyme activity were seen in the range of 1 to 10 mM Mg2+. Magnesium ion concentration acts as an assay for testing the purity of the RNase produced.
Thermal stability and temperature dependence of Pyrococcus abyssi RNase H2
The results should prove the following characteristics of the P.a. RNase H2 enzyme. It should be highly resistant to heat inactivation and ability to tolerate thermocycling conditions. Minimal cleavage efficiency should be achieved around room temperature. Generally, at room temperature, there will no cleavage. Maximum cleavage efficiency should be higher temperatures like 70oC. Tha activity range of the enzyme should be from 50oC- 70oC.
Effect of single base pair mismatches on substrate cleavage by P.a. RNase H2
The sensitivity of the enzyme is tested in this procedure. It is done against a single base mismatch on a series of 30 primer heteroduplex substrates. When the mismatch is at Outside positions “-3 to +1”, there will be little or no effect on cleavage by the enzyme. But when the mismatch at positions “-1” and “0” the effect of the mismatch will be high. The activity of the enzyme in these substrates may have a drop of at least 10-fold.
Characteristics of rhPCR
In this method the requirement of restriction enzyme recognition sequence be located near the 3’-end of the primer would severely limit the use of this method. They hinder the phosphorylation and primer running in the PCR setup.
When using Type I RNase H enzyme will not cleave ta substrate having a single RNA residue. At least 3 consecutive RNA residues are required and 4 for a high-level catalytic activity. This adds cost and complexity of the synthesis of primer. Also, this increases the susceptibility of the template for degradation. When this primer is cleaved it has two or more RNA residues which can inhibit primer extension.
The specificity of the RNase is really high that a mismatch in a DNA: RNA base pair completely prevents amplification.
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. This makes the applicability of the RNase enzyme even for reactions requiring an extended number of cycles and a sustained initial incubation at 95°C.
The reaction efficiency of the RNase enzyme remained unchanged even after 80 cycles. This indicates the enzyme’s thermal stability is sufficient to remain active throughout the range of use expected for all PCR applications.
P.a. RNase H2 has sufficient activity at 50°C to support rhPCR, permitting the reaction to be performed throughout a broad temperature range. Even at low-level concentrations of Mg2+, the enzyme is highly active. Enabling rhPCR to be performed at all magnesium concentrations.
Cleavage occurs at a riboA:deoxyU base pair, allowing the uracil- N-glycosylase (UNG) sterilization method to be used in rhPCR assays. Regardless of the fact that the reaction rates are slightly affected by the identity of the RNA base and the flanking DNA sequence, same concentration of the enzyme and set of reaction conditions generally can be used irrespective of the sequence of the target. Non-specific hydrolysis of the RNA linkage cannot lead to primer activation. Water catalyzed hydrolysis and enzymatic cleavage are the two types of con-specific hydrolysis reaction that is capable of happening inside the rhPCR system. In either case, primer extension remains blocked.
In case of samples containing a significant amount of contamination with single-stranded ribonucleases, inhibitors such as human placental RNase inhibitor can be included in the reaction mixture as RNase H enzymes are not affected by the inhibitors.
Type II RNase H enzymes are capable of cleaving substrates when there is an RNA: DNA base pair mismatch, but the rate at which the reaction happens is comparatively slower than the corresponding perfect match. The rate of the reaction is decreased by about 10-fold. Even in assays which are prone to side reactions the formation of primer-dimers is prevented. This characteristic of the rhPCR can be applied in case of multiplex assays. The specificity of the assay with respect to amplification of homologous sequences is far greater than can be achieved by PCR using unmodified primers. This high degree of specificity should be very useful for the detection of low levels of heterologous DNA in xenogeneic transplant models. In rhPCR, the discrimination between variant alleles relies on differential amplification of the matched and mismatched target sequences.