The Genotying Method Based On The Specific Cleavage Of Rnase Hⅱfrom Chlamydia Pneumoniae

SNP is the single nucleotide polymorphism stemmed from single base mutation of the genome DNA. It has become apparent that SNPs will represent the next generation of markers. Genotyping SNPs of human DNA is a potentially effective way of determining disease susceptibility or drug sensitivity among individuals. Numerous methods have been introduced to genotyping SNPs, However, these methods are usually technically difficult or high cost. For the sake, a novel, accurate, high-throughput, and automation accessible method is preferable.RNase H enzymes are ribonucleotide-specific endonucleases that cleave the RNA portion of RNA-DNA/DNA or RNA/DNA duplex. The activities of RNase H depend on a divalent positive ion, releasing oligo-ribonucleotides with 3'-OH and 5'-phosphate ends. Based on the difference in the amino acid sequences and specificity of enzyme activity, RNase H is classified into two major families, Type 1 RNase H and Type 2 RNase H.Chlamydia pneumoniae does not have genes encoding Type 1 RNase H, but has two genes encoding Type 2 RNase H with different sequences (CP0654 and CP0782) by analyzing the whole genome sequence. The enzymes which are encoded by CP0654 and CP0782 are named as CpRNase HII and CpRNase HIII respectively. We cloned CpRNase HII and CpRNase HIII gene. Recombinant CpRNase HII and CpRNase HIII were expressed and purified. There are different activities between CpRNase HII and CpRNase HIII. CpRNase HII cleaves 12-bp RNA/DNA substrate at multiple sites, while CpRNase HIII only one cleavage site. The most optical concentrations of Mg²⁺ and Mn²⁺ for CpRNase HII cleavage on 12-bp RNA/DNA substrate are 1 mM and 0.1 mM respectively, while the most optical concentrations of Mg²⁺ and Mn²⁺ for CpRNase HIII cleavage are both 10 mM. CpRNase HIII could not cleavage 21-bp chimeric DNA-rN1-DNA/DNA substrates (rN1=one ribonucleotide), while CpRNase HII could cleave them without ribonucleotide preference. The most optical concentration of Mg²⁺ for CpRNase HII cleavage on DNA-rN1-DNA/DNA substrates is 10 mM.Synthetic DNA-rN1-DNA probes (rN1=one ribonucleotide) and complementary oligonucleotides were prepared to generate DNA-rN1-DNA/DNA duplexes, each containing one mismatch at or around the ribonucleotide. The mismatches of duplexes result in slower cleavage rates compared to the matched duplexes. The mismatches located far away from the ribonucleotide have little impact on the cleavage rate of CpRNase HII, yet a greater reduction in cleavage activity is observed for the mismatches located at or adjacent to the ribonucleotide. In conclusion, the impact on the cleavage rate of CpRNase HII, which is imposed by mismatches, is related with the position of mismatches on the DNA-rN1-DNA/DNA duplexes.Ten DNA-rN1-DNA probes and corresponding oligonucleotides were prepared to cover all possible mismatches located at or adjacent to the ribonucleotide. The mismatches at these positions all result in various reductions of CpRNase HII-cleavage rates on DNA-rN1-DNA/DNA duplexes. It was confirmed that the average reduction of cleavage rates of duplexes containing mismatches at ribonucleotide (89.9%) or on the 5'-side of ribonucleotide (86.8%) was more than those on the 3'-side of ribonucleotide (66.9%). The mismatches at the same position of DNA-rN1-DNA/DNA duplexes have different impact on the cleavage rates of CpRNase HII depending on the types of mismatches. These findings may offer further insights into the physical binding and catalytic properties of CpRNase HII–substrate interaction.It has been confirmed that CpRNase HII cleaves the DNA-rN1-DNA/DNA duplex at 5'-side of ribonculeotide (rN1=one ribonucleotide) without ribonucleotide preference. Moreover, the cleavage efficiency of the perfectly matched DNA-rN1-DNA/DNA duplexes is higher than that of the duplexes containing one mismatch at the ribonucleotide. Thus, CpRNase HII can differentiate between one-nucleotide variations on the DNA-rN1-DNA/DNA duplexes.Here, we develop a novel method for SNP genotyping based on specific cleavage properties of CpRNase HII, termed"CpRNase HII-based method". DNA-rN1-DNA fragments are modified with a fluorophore at 5'-end and a quencher at 3'-end to generate chimeric molecular beacons (cMBs), which hybridize with single-stranded DNA (analyte) to be cleaved by CpRNase HII. If cMBs are matched with the target DNA, cMBs are cleaved by CpRNase HII to generate fluorescence. After the first cycle of cleavage, the excessive cMBs would hybridize with the released target ssDNA from the cleavage complex and initiate the second cycle cleavage by CpRNase HII. Thus, further fluorescence is released from the reaction mixture. On the other hand, if cMBs are mismatched with the target, cMBs are not cleaved by CpRNase HII effectively leading to the invalid release of target ssDNA and preventing further cleavage of excessive cMBs in subsequent cycle of reaction as well as the accumulation of the fluorescent signal.CpRNase HII had the ability to detect all kinds of mismatches with high specificity. Moreover, the genotypes were easily distinguishable with 209, 678 and 1680-nt fragments by CpRNase HII-based method. Using CpRNase HII-based method, fluorescence intensity changes and match/mismatch signal ratios were amplified with increased cMBs at the presence of the same amount of target ssDNA, which made SNP genotyping more simple and reliable. We have validated this method with 9 SNPs of HLA gene, which were successfully determined by end-point measurements of fluorescence intensity. The new method is simple and effective, because the design of cMBs is easy and all steps of the genotyping consist of simple additions of solutions and incubation. This method will be suitable for high throughput genotyping.