A simple method for the detection of CYP2C9 polymorphisms: nested allele-specific multiplex polymerase chain reaction.
BACKGROUND
Cytochrome P4502C9 (CYP2C9), a principle drug-metabolizing enzyme is polymorphic in humans and is responsible for important pharmacokinetic and pharmacodynamic variations of CYP2C9 substrates. We developed an allele-specific multiplex polymerase chain reaction (PCR) method for the detection of common CYP2C9 alleles.


METHODS
Genomic DNA was extracted from blood obtained from 40 unrelated healthy Malaysian Indian volunteers. The DNA was subjected to a first PCR that was used to amplify both exons 3 and 7 simultaneously in one reaction tube and a second PCR that was used to detect the polymorphic sites of CYP2C9 alleles using allele-specific primers. Sequencing was performed to validate the test results.


RESULTS
We were successful in amplifying the fragments of interest from the DNA samples. The method was also reproducible and specific. The amplified sequences showed 100% homology to CYP2C9 sequence.


CONCLUSIONS
This is the first nested allele-specific multiplex PCR method reported to allow for the simultaneously detection of five CYP2C9 alleles.
D000483:Alleles; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D065729:Cytochrome P-450 CYP2C9; D004247:DNA / Q000097:blood; D017931:DNA Primers / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005838:Genotype; D006801:Humans; D015636:Magnesium Chloride / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic / Q000235:genetics
D017931:DNA Primers; D015636:Magnesium Chloride; D004247:DNA; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases