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


METHODS
Two 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.


RESULTS
In 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.


CONCLUSIONS
The 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.
Ye
Rong
R
Laboratory of Molecular Biology, Hangzhou Center For Disease Control and Prevention, Hangzhou, Zhejiang, 310006 P. R. China. yerongcn@yahoo.com
Lai
Yuqiao
Y
Pan
Jing-cao
JC
D000293:Adolescent; D000328:Adult; D001483:Base Sequence; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D055815:Young Adult
D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human