engleski

Detection of factor V Leiden by PCR-SSCP using precast Elchrom Scientific GMATM gels

A point mutation in the coagulation Factor V gene (FV:R506Q) called Factor V Leiden is the most common cause of thrombophilia. Since the prevalence of Factor V Leiden in normal population is high, it would be reasonable to perform a simple and cost-effective method for screening the genetic abnormality in population at risk. Here we present a new simple, reproducible and cheap PCR-SSCP method that allows us to identify the carriers of Factor V mutation. Single-Strand Conformation Polymorphism (SSCP) is a mutation detection method based on changes in the secondary structure within a defined ssDNA fragment. The aim of this study was to optimize PCR-SSCP method for detection of Factor V Leiden mutation using the precast GMA™ gels in the Elchrom Scientific SEA 2000 apparatus. We performed PCR-SSCP analysis on 50 whole blood DNA samples previously genotyped by PCR-RFLP method. Exon X of Factor V was amplified using previously described primers and denatured at 95°C during 5 minutes. Denatured PCR products were loaded and run overnight on precast GMA gels in SEA 2000 electrophoresis apparatus. Gels were stained for 30 min with SYBR Gold nucleic stain, destained for 40 min in double distilled water and photographed at 254 nm with Polaroid 667 film. We observed reproducible and uniform band patterns for mutant and wild type variants of Factor V. Our PCR-SSCP results were verified using the restriction pattern after Mnl I digestion of the same PCR product. Results always matched completely, the concordance between both methods was 100 %. Technique has shown to be highly reproducible because of high performance characteristics of GMA gels and constant temperature maintenance by Elchrom Scientific SEA 2000 apparatus. In conclusion, described PCR-SSCP procedure is reliable, time saving and cost effective and should therefore be useful in the routine detection of Factor V Leiden.

factor V Leiden; PCR-SSCP

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