Rapid Detection of the Factor V Leiden Mutation by Real-Time PCR with TaqMan Minor Groove Binder Probes

Individuals heterozygous for the factor V Leiden mutation have a sevenfold higher risk to develop venous thrombosis than wild-type (WT) individuals (1). The allele frequency of factor V Leiden in Europeans is ∼4.4%, and the mutation is currently considered as the most important genetic risk factor for venous thrombosis (2). Various methods have been published to detect the factor V Leiden mutation by real-time PCR using hybridization probes (3)(4)(5) and TaqMan probes (6)(7). Here we describe a robust and convenient procedure for the detection of the factor V Leiden mutation that uses TaqMan probes conjugated to a minor groove binder (MGB) group. TaqMan probes are degraded during amplification by the 5′→3′ exonuclease activity of the polymerase. During degradation of the probe, the fluorescent group is separated from the quencher, leading to increased fluorescence. For allelic discrimination, probes matching the WT and the mutant allele are conjugated to a different fluorescent group. Discrimination between alleles is based on the difference in melting temperature (Δ T m) between the differently labeled match and mismatch probes. For TaqMan assays, relatively long probes are required that remain annealed during the extension phase of the PCR. A 1-bp mismatch in such a probe may lead to a small Δ T m and subsequent difficult allelic discrimination. DNA probes conjugated to a MGB group …