engleski

A new genotyping method for PAI-1 4G/5G polymorphism

Plasminogen activator inhibitor-1 (PAI-1) plays important role in fibrinolysis by inhibiting tissue-plasminogen activator (tPA). Although controversal reports are present, 4G/5G single nucleotide I/D polymorphism in the PAI-1 promoter region seems to be associated with myocardial infarction, thrombosis and development of vascular disease in NIDDM patients (1). Genotyping of this polymorphism is commonly detected with an allele-specific PCR. Thus, our aim was to introduce a new, quick and easy method for genotyping PAI-1 4G/5G polymorphism. The method was based on PCR amplification followed by Single Stranded Conformation Polymorphism (SSCP) analysis and electrophoresis on the precast GMA˘â gels in the Elchrom Scientific SEA 2000 apparatus. We analysed 50 whole blood DNA samples of healthy volunteers. DNA was extracted by standard methodology and two PCR fragments (188 and 189 bp respectively) were amplified using previously described primers (1). SSCP analysis was performed by mixing 6 Ľěl of PCR product with 14 Ľěl of formamide containing 10 mM NaOH, 0.1% bromphenol blue, denatured at 95˘ŞC for 5 min and quickly cooled on ice. 10 Ľěl of the mixture were electrophoresed over night (17 hours) with constant buffer temperature (30 mM TAE buffer) at 14˘ŞC and 2.5 V/cm. The electrophoresed bands were visualized with SYBR Gold after 1 hour of staining and photographed at 254 nm with Polaroid 667 film. The method allowed us to determine the genotype of all tested samples. Homozygous and heterozygous genotypes were concordant to previously obtained controls. Our PCR-SSCP results showed uniform and reproducible band patterns achieved with constant temperature conditions of the SEA 2000 apparatus and high performance characteristics of GMA˘â gels. The method showed to be reliable and cost effective and therefore could be used in laboratories since it is very easy to perform. This analysis could become more important if the PAI-1 genotype is confirmed to be a risk factor for myocardial infarction.

genotyping method; plasminogen activator inhibitor-1; 4G/5G polymorphism

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