RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers
Single nucleotide polymorphisms (SNPs) provide a great opportunity for the study of human disease and bacterial drug resistance. However, many SNP typing techniques require dedicated instruments and high cost. Here, we develop a novel method for SNP genotyping based on specific cleavage properties of RNase HII from Chlamydia pneumoniae (CpRNase HII), termed the "CpRNase HII-based method." CpRNase HII cleaves the DNA-rN(1)-DNA/DNA duplex at the 5'-side of the ribonucleotide (rN(1) = one ribonucleotide). Moreover, the cleavage efficiencies of the perfectly matched DNA-rN(1)-DNA/DNA duplexes are higher than those carrying a mismatched ribonucleotide. DNA-rN(1)-DNA fragments are modified with a fluorophore at the 5'-end and a quencher at the 3'-end to generate molecular beacons (MBs), which hybridize with single-stranded DNA (analyte) to be cleaved by CpRNase HII. As perfectly matched duplexes can be cleaved efficiently and mismatched duplexes cannot, CpRNase HII-catalyzed reactions can differentiate between one-nucleotide variations on the DNA-rN(1)-DNA/DNA duplexes. We have validated this method with nine SNPs of the HLA gene, which were successfully determined by endpoint measurements of fluorescence intensity. The new method is simple and effective, because the design of MBs is easy, and all steps of the genotyping consist of simple additions of solutions and incubation. This method will be suitable for large-scale genotyping.