Electrophoretic patterns for multiplex FV and FII PCR. The PCR mixture in a final volume of 50 μL consisted of 1 μg genomic DNA, PCR buffer (16.6 mmol/L amonium sulphate, 67 mmol/L Tris-HCl, pH 8.8, 6.7 mmol/L magnesium chloride, 67 μmol/L Na2EDTA, 170 μg bovine serum albumin per mL, 10 mmol/L β-mercaptoethanol), 400 μmol/L of each desoxynucleotide triphosphate, 30 pmol of each FV primer, 10 pmol of each FII primer, and 2 U Taq polymerase. Thermocycling conditions are 94°C (1 minute), 58°C (1 minute), 72°C (2 minutes) for 40 cycles. Fifteen microliters of PCR product is digested with 15 U HindIII restriction enzyme. The restricted products are separated by electrophresis through a 2% agarose gel stained with ethidium bromide and directly visualized under UV light. The smallest restricted fragments (32 and 23 bp) are not visible on the gel. Lane 1, size marker (1-kb ladder); lane 2, undigested PCR products; lanes 3 through 8, digested PCR products. UD, undigested PCR products; N, normal allele; m, mutated allele.
