AN ALTERNATIVE METHOD FOR IDENTIFYING THE FACTOR-V GENE LEIDEN MUTATION

The recently described point mutation of nucleotide 1691 of the factor V gene is responsible for the factor V resistance to a cleavage with an activated protein C (APC-resistance). Despite the high sensitivity and specificity of the APC-resistance test as a screening method for t he detection of the factor V APC-resistance, there is still a necessit y to develop a simple, cheap and accurate DNA-based assay. Herein, two different ASO-PCR methods were used for the detection of the Leiden m utation. The first involved a direct ASO-PCR with a consensus FV N1 pr imer and a sequence specific primer for the 1691 bp ''G'' normal allel e (FV G1) or a specific primer for the 1691 bp ''A'' mutant allele (FV A1). The second method consisted of direct ASO-PCR by using the conse nsus FV N1 primer and one of two primers with an additional T-->G mism atch at the penultimate position from the 3'-end. One primer was speci fic for the 1691 bp ''G'' normal allele (FV G2) and the other was spec ific for the 1691 bp ''A'' mutant allele (FV A2). These permit clear a nd easy distinctions between homozygous normal and heterozygous and ho mozygous mutant probands. We also tested a T-->A mismatched primer to compare our method with that recently reported by Bellisimo et al. We found that within a large range of PCR conditions, T-->G mismatched ol igonucleotides discriminated better than T-->A mismatched between thre e factor V 1691 position genotypes. We therefore recommend our method for the screening of a single 1691 G-->A nucleotide mutation in the fa ctor V gene. Copyright (C) 1997 Elsevier Science Ltd.