As a starting material for PCR, trisodium citrate anticoagulated whole blood, was used. A 267 bp fragment containing the factor V polymorphic site and a 345 bp fragment containing the factor II polymorphic site, were amplified simultaneously, using primers previously described: 5'-TGCCCAGTGCTTAACAAGACC-3 ', 5'-TGTTATCACACTGGTGCTAA-3' for factor V; and 5'-TCTAGAAACAGTTGCCTGGC-3', 5'-ATAGCA CTG GGAGCATTGAAGC-3' for factor II [1,2]. The PCR analyses were performed in a 25 mL final volume containing 50 mM KCl, 100 mM Tris-HCl, pH 9.0, 0.1% Triton X-100, 200 mM dNTPs, and varying amounts of MgCl2, four primers, DNA polymerase (Promega, Madison, WI, USA) and blood. The reaction mixtures were overlaid with a drop of mineral oil and the following cycling conditions were applied: 5 cycles consisting of 98°C for 3 minutes and 55°C for 3 minutes. After that, Taq polymerase was added at 50°C and after 5 minutes, 35 cycles consisting of 94°C for 1 minute, 57°C for 1 minute and 72°C for 1 minute were applied. Final polymerization was at 72°C for 10 minutes.

Ten mL of amplified samples were digested overnight, at 37°C, with the MnlI and HindIII enzymes (Biolabs, New England, USA). Digestion products were analyzed by electrophoresis on 10% polyacrylamide gels followed by silver staining of the gels [7].