High throughput genotyping for the detection of a single nucleotide polymorphism in NAD(P)H quinone oxidoreductase (DT diaphorase) using TaqMan probes.
OBJECTIVE
The two electron reduction of quinones to hydroquinones by NAD(P)H quinone oxidoreductase (NQO1) plays an important role in both activation and detoxification of quinone and similarly reactive compounds. A single nucleotide polymorphism at exon 6 leads to an amino acid change at codon 187 from proline to serine. The variant allele has been associated with decreased NQO1 enzyme activity and increased cancer risks. The aim of this study was to develop a rapid genotyping procedure for epidemiological and clinical research into the potential biological and toxicological implications associated with this genetic polymorphism.


METHODS
A high throughput genotyping method using fluorogenic probes has been developed to screen this single nucleotide polymorphism. This assay utilises the 5' nuclease activity of Taq polymerase in conjunction with fluorogenic TaqMan probes. The TaqMan genotyping procedure was validated by a restriction fragment length polymorphism method and direct sequencing.


RESULTS
This method can be used for the rapid screening of known polymorphisms in large populations. In a population of 143 unrelated individuals, Pro/Pro (wildtype), Pro/Ser (heterozygous), and Ser/Ser (mutant) genotypes were 69.2%, 26.6%, and 4.2%, respectively.


CONCLUSIONS
This genotyping method is highly accurate and could be applied to automated large scale genotyping studies.
Shi
M M
MM
Genomic Pathology Laboratory, Parke-Davis Pharmaceutical Research, Warner-Lambert Company, Ann Arbor, MI 48105, USA. michael.shi@wl.com
Myrand
S P
SP
Bleavins
M R
MR
de la Iglesia
F A
FA
D000483:Alleles; D005456:Fluorescent Dyes; D005838:Genotype; D006801:Humans; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D019914:Taq Polymerase
D005456:Fluorescent Dyes; D016660:NAD(P)H Dehydrogenase (Quinone); D019914:Taq Polymerase