Detection of single nucleotide polymorphism by RNase H-cleavage mediated allele-specific extension method

Allele-specific extension is one of the most powerful and promising tools for the detection of single nucleotide polymorphisms. However, false negative signals are easily produced using this method especially for G-T and C-A mismatches. Here, we describe a novel and efficient SNP detection method based on RNase H-cleavage mediated allele-specific extension (RCAE). The technique is based on the cleavage characteristic of Chlamydia pneumoniae RNase HII (CpRNase HII) and allele-specific extension. The CpRNase HII cleavage efficiencies of DNA-rN1-DNA/DNA duplexes (rN1=one ribonucleotide) are 4.2- to 11.1- fold greater than those of mismatched duplexes containing mismatches on the 5'-side of the hydrolyzed phosphodiester. In this design, DNA-rN1-DNA probes containing polymorphic base on the 5'-side of the hydrolyzed phosphodiester for each selected SNP are annealed with the DNA samples individually to generate the matched or mismatched duplex. The matched duplex can be cleaved by CpRNase HII and one of the cleaved products serves as a primer for allele-specific extension, while allele extension is not carried out for the mismatched duplex. Thus, RCAE differentiates between one-nucleotide variations in DNA-rN1-DNA/ DNA duplexes. The method was validated by typing SNPs in HLA gene, demonstrating it can be used to accomplish accurate and parallel SNP genotyping.