Rapid detection of the factor V Leiden mutation by real-time PCR with TaqMan minor groove binder probes.

Troponin-tropomyosin interactions. Fluorescence studies of the binding of troponin, troponin T, and chymotryptic tropo-nin T fragments to specifically labeled tropomy-osin. To the Editor: Individuals heterozygous for the factor V Leiden mutation have a seven-fold higher risk to develop venous thrombosis than wild-type (WT) individuals (1). The allele frequency of factor V Leiden in Europeans is ϳ4.4%, 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 Lei-den mutation by real-time PCR using hybridization probes (3–5) and Taq-Man probes (6, 7). Here we describe a robust and convenient procedure for the detection of the factor V Lei-den mutation that uses TaqMan probes conjugated to a minor groove binder (MGB) group. TaqMan probes are degraded during amplification by the 5Ј33Ј exo-nuclease 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 (⌬T m) between the differently labeled match and mismatch probes. For TaqMan assays, relatively long probes are required that remain an-nealed during the extension phase of the PCR. A 1-bp mismatch in such a probe may lead to a small ⌬T m and subsequent difficult allelic discrimination. DNA probes conjugated to a MGB group form hyperstabilized duplexes with complementary DNA by folding of the MGB group into the duplex, giving a higher T m (8, 9). The conjugation of a MGB group therefore allows the design of shorter probes with an increased specificity attributable to increased mismatch discrimination (9). For detection of the factor V Lei-den mutation, we designed MGB probes with a length of 17 bases, which is Ͼ25% shorter than the length (23–24 bases) of previously published TaqMan probes for factor V Leiden detection (6, 7) without a MGB group. In addition, the probes were designed with the mutation site located in the MGB region to achieve the maximum ability to discriminate alleles (9).