RNA-primed allele-specific PCR

Abstract

RNA/DNA primer pairs and two polymerases were used to efficiently amplify DNA sequences using the conventional polymerase chain reaction (PCR). The reaction required the use of both DNA polymerase and reverse transcriptase during each thermal cycle and formed a double-stranded DNA in which one terminus was an RNA/DNA hybrid. Because there is a higher sensitivity of the DNA polymerase to the mismatch at the 3′-end in the RNA/DNA hybrid duplex than in the DNA/DNA duplex, the RNA-primed PCR reveals much better specificity in the allele-specific PCR to detect single-nucleotide mutation.