Genotyping of cytochrome P450 2D6*3 and *4 mutations using conventional PCR.
Cytochrome P450 (CYP450) mixed-function mono-oxygenases, consisting of more than 30 enzymes, are responsible for the metabolism of a large number of drugs and metabolites. With the rapid advances in the human genome project, the role of genetic polymorphism in drug metabolism may become an important adjunct for rational drug therapy, and for the explanation of drug toxicity and interactions. This preliminary study modified a previously described procedure for genotyping CYP2D6*3 and *4. An additional step included uracil-DNA glycosylase for the prevention of "carry-over" contamination. DNA was extracted from peripheral blood using PureGene DNA Isolation kit. CYP2D6*3 and *4 sequences were amplified by PCR, followed by digestion with restriction endonuclease Msp1 and Mva1, respectively. Resulting fragments were analyzed by electrophoresis and visualized by ethidium bromide staining. Poor metabolizers of *3 mutation showed 168-, 82- and 20-bp bands, while those of *4 showed a single 355-bp band. Using these protocols, 22 individuals were genotyped, showing the following prevalence for *3 and *4: 0 and 3, respectively-comparable to those of the general population. This method provides a reliable means of genotyping CYP2D6*3 and *4.
Schur
B C
BC
Department of Pathology, Medical College of Wisconsin, P.O. Box 26509, Milwaukee, WI 53226, USA.
Bjerke
J
J
Nuwayhid
N
N
Wong
S H
SH
D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics*; D004247:DNA / Q000097:blood*; D045647:DNA Glycosylases; D004262:DNA Restriction Enzymes; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004996:Ethidium; D005838:Genotype; D006801:Humans; D009154:Mutation / Q000235:genetics*; D009699:N-Glycosyl Hydrolases; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics*; D015203:Reproducibility of Results; D051981:Uracil-DNA Glycosidase
D004247:DNA; D019389:Cytochrome P-450 CYP2D6; D004262:DNA Restriction Enzymes; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D004996:Ethidium