Polymerase chain reaction with confronting two-pair primers for polymorphism genotyping

Abstract

A novel PCR method using confronting two-pair primers, named PCR-CTPP, is introduced to detect a single nucleotide polymorphism (base X or Y). One primer for the X allele is set to include X' at the 3' end (antisense), where X' is the antisense of X, with the counterpart sense primer upstream. For the Y allele, a sense primer including Y at the 3' end is set, with the antisense primer downstream. One common band and one specific band for each allele are amplified, which allows genotyping directly by electrophoresis. This method is exemplified by application to the polymorphisms of beta-adrenoceptor 2 and interleukin 1B. It is simpler than PCR-RFLP (restriction fragment length polymorphism), which requires incubation with a restriction enzyme, and is suitable for genotyping in studies of genetic epidemiology involving hundreds of samples.