engleski

PCR-SSCP method for detection of the CYP2D6 gene *3 and *4 mutations

The metabolism of many drugs and xenobiotics involves the oxidative processes via the cytochrome P450 enzymes of the microsomal liver fraction. One isoform of the cytochrome P450 enzyme group is CYP2D6, which metabolizes more than 25% of the most commonly used drugs. The most frequent alleles of the CYP2D6 isoform are CYP2D6*3 and CYP2D6*4, and the most common method of their detection is PCR-RFLP. The aim of the study was to optimize the PCR-SSCP method of detection of CYP2D6*3 and *4 mutations using the precast GMAÔ gels in the Elchrom Scientific SEA 2000 apparatus. The method is based on change in the secondary structure within a defined single strand DNA fragment. PCR-SSCP analysis was performed on 50 whole blood samples previously genotyped by the PCR-RFLP method. The results showed reproducible and uniform band patterns for mutant and wild type variants of CYP2D6*3 and *4 alleles. Each sample previously genotyped by the method of PCR-RFLP was confirmed by the method of PCR-SSCP (100% concordance). The technique is reliable, simple and cost effective due to the high resolution of GMAÔ gels and constant temperature maintained by the Elchrom Scientific SEA 2000 apparatus.

PCR-SSCP; polymorphism; cytochrome P450 2D6

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