Rapid Single-Tube Genotyping of the Factor V Leiden and Prothrombin Mutations by Real-Time PCR Using Dual-Color Detection

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Abstract
Patients carrying the G1691A mutation in the factor V gene (factor V Leiden) have been demonstrated to be at risk for venous thromboembolism. A second polymorphism also associated with hereditary thrombophilia was identified in the prothrombin gene (G20210A). Because of the high prevalence of these two mutations (5–10% for G1691A and 2–4% for G20210A) in the Caucasian population, there is growing demand for rapid, reliable, and simple methods for combined detection of both point mutations. Numerous PCR-based assays have been described for the detection of each of the mutations separately (1)(2)(3)(4) as well as in combination by multiplex PCR analysis (5)(6)(7)(8)(9)(10) and other single-tube alternatives [see, for example, Ref. (11)]. All of these methods, however, are time-consuming and require multiple manual steps such as restriction length polymorphism analysis, electrophoresis, and hybridization with specific oligonucleotide probes.