Rapid detection of CYP2C9*3 alleles by real-time fluorescence PCR based on SYBR Green.
CYP2C9 catalyzes the metabolism of important drugs such as phenytoin, S-warfarin, tolbutamide, losartan, and nonsteroidal anti-inflammatory drugs. A functional polymorphism of the CYP2C9 gene has been described. The single-base mutation of A1061C (Ile359Leu) in the CYP2C9 gene termed CYP2C9*3 was found at a frequency of about 2.1% in Japanese. We developed a rapid mutation analysis method for detecting the CYP2C9*1 genotype. This method is a marriage of two emerging technologies: allele-specific amplification primers for target DNA and a new double-stranded DNA-selective fluorescent dye, SYBR Green. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. We applied this procedure to DNA extracted from the blood of healthy Japanese volunteers. The CYP2C9 wild-type CYP2C9*1/CYP2C9*1 and heterozygous CYP2C9*1/CYP2C9*3 genotypes of the CYP2C9 alleles detected by the assay were consistent with the results obtained from restriction enzyme cleavage. No genotype of CYP2C9*3/CYP2C9*3 was found in these samples. Using plasmid DNA containing a point mutation of CYP2C9*3 as template, the assay separated the three genotypes. We conclude that this simple, rapid, and inexpensive procedure is applicable to routine high-throughput assays.
Hiratsuka
M
M
Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, 980-8574, Japan.
Agatsuma
Y
Y
Mizugaki
M
M
D000483:Alleles; D019943:Amino Acid Substitution; D001189:Aryl Hydrocarbon Hydroxylases; D003001:Cloning, Molecular; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004247:DNA / Q000097:blood; D005456:Fluorescent Dyes; D005838:Genotype; D006720:Homozygote; D006801:Humans; D007564:Japan; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics*
D005456:Fluorescent Dyes; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase