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) polymorphism was used as an example. Two sets of four primers were applied for PCR-CTPP; the first set with different melting temperatures (Tms), and the second with similar Tms. 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 Tms 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.
Hamajima
Nobuyuki
N
Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, 1-1 Kano-koden, Chikusa-ku, Nagoya 464-8681, Japan. nhamajim@aichi-cc.jp
Saito
Toshiko
T
Matsuo
Keitaro
K
Tajima
Kazuo
K
D017931:DNA Primers; D005838:Genotype; D006801:Humans; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic
D017931:DNA Primers; D016660:NAD(P)H Dehydrogenase (Quinone)