OBJECTIVETo develop a single-tube fluorescent bidirectional PCR method to detect the 609C/T polymorphism of NAD(P)H: quinone oxidoreductase 1 (NQO1) gene.

METHODSTwo primers of NQO1 gene C609T locus were designed. Using these primers, a SYBR Green I fluorescent bidirectional PCR, combined with melting curve analysis of the PCR products, were optimized to differentiate the 609C/T polymorphisms in 191 samples of human genomic DNA. The accuracy of the fluorescent bidirectional PCR was validated by the classical method of PCR-restriction fragment length polymorphism(RFLP) in 62 of these 191 samples.

RESULTSIn the 62 samples, the genotypes determined by the fluorescent bidirectional PCR were 100% consistent with the ones by the PCR-RFLP. The frequencies of genotypes of homozygous wild-type (CC), heterozygous (CT), and homozygous mutant (TT) were 28%, 50%, and 22%, respectively, in the 191 samples.

CONCLUSIONThe single-tube fluorescent bidirectional PCR method established here provides a simple, rapid, accurate and inexpensive assay to determine the 609C/T polymorphism of NQO1 gene. The assay is suitable to detect the single nucleotide polymorphism in large-scale samples.

Adolescent ; Adult ; Base Sequence ; Female ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; NAD(P)H Dehydrogenase (Quinone) ; genetics ; Polymerase Chain Reaction ; methods ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; genetics ; Reproducibility of Results ; Young Adult