engleski

Detection of CYP2D6 A and B mutations by SSCP using the Precast GMATM gels in the Elchrom Scientific sea 2000 apparatus

Single-Strand Conformation Polymorphism (SSCP) is a method for detection of known as well as unknown mutations. It is based on changes in the secondary structures in a defined single-stranded DNA fragment caused by a change in sequence. These changes in structure alter the fragment mobility during the gel electrophoresis. As one of the easiest and very cost effective methods with high mutation detection rates (more than 95 percent for some genes) it is suitable for screening the large number of samples. Eventhough SSCP is quick and easy to perform, a major disadvantage is related to its reproducibility. There are several conditions important for consistent detection of SSCP bands: gel composition, PCR product length and dilution, loading volume, denaturants, loading buffer, running buffer, running voltage, and the most important - temperature. Electrophoresis should be performed at low and constant temperature in order to maintain particular conformation of ssDNA fragments. The aim of this study was to design a rapid and simple method for detection of CYP2D6 A and B mutations by SSCP using the precast GMA™ (Gene Mutation Analysis) gels in the Elchrom Scientific SEA 2000 apparatus. We performed CYP2D6 A and B genotype SSCP analysis on 30 whole blood DNA samples previously genotyped by PCR-RFLP method. CYP2D6 exon IV (B allele) and exon V (A allele) were amplified and denaturated by heating at 95 C for 5 minutes. The electrophoresis was performed with GMA™ ready to use gels (optimal separation range of 150 - 300 bp) in SEA 2000 electrophoresis apparatus specially suited with heat exchange element and laminar buffer flow pump that maintains even temperature over the whole gel surface. The electric field strength was 45 V and buffer temperature was maintained at 8 C. The electrophoresis was run overnight (18 hours). Gels were stained with SYBR Green II for 30 min, destained in double distilled water for 40 minutes and photographed at 254 nm with Polaroid 667 film. We observed reproducible and uniform band patterns for mutant and wild type variants of CYP2D6 A and B genotypes. Our findings were consistent with previously genotyped samples. This suggests that the presently known CYP2D6 mutations can be distinguished by PCR-SSCP analysis using the precast GMA™ gels in the Elchrom Scientific SEA 2000 apparatus. This method is reliable and more suitable than previously used PCR-RFLP for identification of known mutations, and it simultaneously screens for new mutations.

CYP2D6 A and B mutations; SSCP; GMA

nije evidentirano