Factor V Leiden genotyping using real-time fluorescent polymerase chain reaction

A fluorogenic probe-based PCR assay (Taqman; Perkin Elmer corp/Applied Biosystems, Foster City, CA, USA) was used for the detection of Factor V Leiden, a point mutation in the factor V gene (G1691A) that is the most common inherited risk factor for Deep Vein Thrombosis. This assay allows for the direct detection of specific PCR products within minutes of completion of the PCR by monitoring the increase in fluorescence of a dye-labelled oligonucleotide probe. Two dye-labelled probes are used in this allelic discrimination assay, one probe for each allele in the two-allele system. Each probe consists of an oligonucleotide with a 5'-reporter dye and a 3'-quencher dye. Tet (6-carboxy-4,7,2',7'-tetrachloro-fluorescein) is covalently linked to the 5'-end of the probe for the detection of allele 2 (wild-type). Fam (6-carboxy-fluorescein) is covalently linked to the 5'-end of the probe for detection of allele 1 (mutant). Each of the reporters is quenched by Tamra (6-carboxy-N,N,N', N'-tetramethylrhodamine) attached via a linker arm located at the 3'-end of each probe. The two probes were complementary to a 24-base sequence at the factor V Leiden mutation site, but differing in the 5'-labelled reporter dye and the nucleotide opposite the mutation site (C vs T). Wild-type and factor V Leiden alleles were differentiated in highly purified DNA and crudely purified DNA specimens. The assay was successfully applied to genomic DNA from leukocytes isolated from whole blood. The factor V status of 120 patients as determined by this method was in complete concordance with a standard PCR-based assay and clearly discriminated between healthy wild-type (+/+), factor V Leiden homozygote (-/-) and heterozygote (+/-) carriers.