Competitive amplification and unspecific amplification in polymerase chain reaction with confronting two-pair primers

Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is an inexpensive, time-saving genotyping method that is applicable for most single nucleotide polymorphisms. To date, we have applied PCR-CTPP successfully for the genotyping of more than 30 polymorphisms. This paper demonstrates the differences in DNA amplification among different annealing temperatures of PCR-CTPP with given melting temperatures for four primers. The NQO1 C609T (Pro187Ser) polymorphisin was used as an example. Two sets of four primers were applied for PCR-CTPP; the first set with different melting temperatures (Tins), and the second with similar Tins. The comparisons with one-pair primer PCR (allele-specific PCR) revealed that PCR-CTPP amplified DNA more specifically than allele-specific PCR. The primers with different Tms caused competitive DNA amplification for heterozygous genotype. Four primers with similar Tins amplified both alleles unspecifically at a lower annealing temperature, while the same DNA samples were correctly genotyped under an optimal annealing temperature. These findings are unique for PCR-CTPP, and important characteristics when the primers and annealing temperatures in PCR-CTPP are designed. The knowledge of these characteristics will extend the applicability of PCR-CTPP for polymorphism genotyping.