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 the detection of the factor V APC-resistance, there is still a necessity to develop a simple, cheap and accurate DNA-based assay. Herein, two different ASO-PCR methods were used for the detection of the Leiden mutation. The first involved a direct ASO-PCR with a consensus FV N1 primer and a sequence specific primer for the 1691 bp "G" normal allele (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 consensus FV N1 primer and one of two primers with an additional T-->G mismatch at the penultimate position from the 3'-end. One primer was specific for the 1691 bp "G" normal allele (FV G2) and the other was specific for the 1691 bp "A" mutant allele (FV A2). These permit clear and easy distinctions between homozygous normal and heterozygous and homozygous 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 oligonucleotides discriminated better than T-->A mismatched between three factor V 1691 position genotypes. We therefore recommend our method for the screening of a single 1691G-->A nucleotide mutation in the factor V gene.
Lewandowski
K
K
Department of Haematology, Karol Marcinkowski University of Medical Sciences, Poznań, Poland.
Rozek
M
M
Zawilska
K
K
Markiewicz
W T
WT
D000483:Alleles; D004247:DNA / Q000032:analysis; D005165:Factor V / Q000235:genetics*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results
C095381:factor V Leiden; D005165:Factor V; D004247:DNA