Improvement of single nucleotide polymorphism genotyping by allele-specific PCR using primers modified with an ENA residue.

When we placed an ENA residue into primers at the 3′ end, or the n-1, n-2, or n-3 position, which included a single nucleotide polymorphism (SNP) site at the 3′ end, only primers containing the ENA residue at the n-2 position were read by Taq DNA polymerase for amplification. The use of the ENA primers avoided the generation of undesired short products, which are thought to be derived from primer–dimers. A greater discrimination of the SNP site by these primers containing the ENA residue was observed compared with that of the corresponding unmodified DNA primers that are often used for allele-specific polymerase chain reaction (AS–PCR). This improvement is probably due to the difficulty of incorporating a nucleotide into the mismatched ENA primer by Taq DNA polymerase in the modified primer–template duplex. These results demonstrate that ENA primer-based AS–PCR would enable a rapid and reliable technique for SNP genotyping.