engleski

A new factor V Leiden PCR-SSCP detection method

Factor V Leiden mutation is the most common cause of thrombophilia. The overall prevalence of Factor V Leiden in healthy population is rather high, and it is therefore necessary to develop and introduce a simple and cost-effective method for screening this genetic abnormality in population at increased risk. We herein present a new, simple, reproducible and cheap PCR-SSCP method that allows us to identify the carriers of Factor V mutation. Our PCR-SSCP method for detection of Factor V Leiden mutation was optimised using the precast GMA™ gels in the Elchrom Scientific SEA 2000 apparatus. We performed PCR-SSCP analysis on 109 whole blood DNA samples previously genotyped by PCR-RFLP method using the Mnl I restriction endonuclease. Exon X of Factor V was amplified using previously described primers and denatured for 5 minutes at 95°C. 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 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. Genotype frequencies were 92.7, 7.3 and 0 for wild type, heterozygotes and homozygotes respectively. Allele frequencies were 96.3 for wild type and 3.7 for mutated allele. The reported frequencies were consistent with those reported in the literature for population of whites. Our PCR-SSCP results were verified using the restriction pattern after Mnl I digestion of the same PCR product. The concordance between both methods was 100%. Technique is highly reproducible because of the high performance characteristics of GMA™ gels and constant temperature maintenance by Elchrom Scientific SEA 2000 apparatus. In conclusion, we recommend herein presented PCR-SSCP procedure as a highly reliable, time saving and cost effective Factor V Leiden detection method.

Factor V Leiden; thrombophilia; PCR-SSCP

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