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PCR-SSCP genotyping of CYP2D6 3 and 4 mutations by precast Elchrom Scientific Gma™ gels

Single-Strand Conformation Polymorphism (SSCP), a detection method for known and unknown mutations is based on changes in the secondary structures within a defined ssDNA fragment. These changes alter the fragment electrophoretic mobility. Electrophoresis should be performed at low and constant temperature in order to maintain particular conformation of ssDNA fragments. The reproducibility of the SSCP is its major disadvantage. Many conditions influence on consistent detection of SSCP bands, however temperature is one of the most important. The aim of this study was to optimize PCR-SSCP method for detection of CYP2D6 3* and 4* mutations using the precast GMA™ gels in the Elchrom Scientific SEA 2000 apparatus. PCR-SSCP analysis was performed on 30 whole blood DNA samples previously genotyped by PCR-RFLP method. Exon IV of CYP2D6 (4* allele) and exon V (3* allele) were amplified and denatured at 95C during 5 minutes. The electrophoresis was performed with GMA™ ready to use gels in SEA 2000 electrophoresis apparatus that is specially suited with heat exchange element and laminar buffer flow pump which maintains uniform temperature over the whole gel surface. Applied electric field was 45 V and buffer temperature was 8C during the overnight run (18 h). Gels were stained for 30 min with SYBR Green II, destained for 40 min in double distilled water and photographed at 254 nm with Polaroid 667 film. The results showed reproducible and uniform band patterns for mutant and wild type variants of CYP2D6 3* and 4* genotypes. PCR-SSCP findings were consistent with genotyped samples by PCR-RFLP. This suggests that the presently known CYP2D6 mutations can be distinguished by PCR-SSCP analysis. Technique is highly reproducible because of high performance characteristics of GMA™ gels and constant temperature maintenance by Elchrom Scientific SEA 2000 apparatus. It can be concluded that the method is time saving and cost effective; reliable and suitable for identification of known mutations, furthermore it simultaneously screens for new mutations in population.

PCR-SSCP; Genotyping; CYP2D6 3 and 4 mutations; Elchrom Scientific Gma™ gels

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