Rapid detection of CYP2C9*3 alleles by real-time fluorescence PCR based on SYBR green

Masahiro Hiratsuka, Yasuyuki Agatsuma, Michinao Mizugaki

Research output: Contribution to journalArticlepeer-review

45 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)357-362
Number of pages6
JournalMolecular Genetics and Metabolism
Volume68
Issue number3
DOIs
Publication statusPublished - 1999 Nov

Keywords

  • Allele-specific amplification
  • CYP2C9
  • Genetic polymorphism
  • P450
  • PCR
  • SYBR Green

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Endocrinology

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