[Establishing a single-tube fluorescent bidirectional PCR method to detect the 609C/T polymorphism of NQO1 gene]
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Published on: September 25, 2018
Rong Ye1, Yuqiao Lai, Jing-cao Pan
1Laboratory of Molecular Biology, Hangzhou Center For Disease Control and Prevention, Hangzhou, Zhejiang, 310006 P. R. China. yerongcn@yahoo.com
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.
Conclusion:
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.
