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

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. Recently, a new detection methodology was introduced on high-speed thermal cyclers based on real-time PCR analysis followed by hybridization of amplicon-specific oligonucleotides with adjacent fluorophores capable of fluorescence resonance energy transfer (LightCyclerTM; Roche Molecular Biochemicals). Probes, labeled with two different fluorescent molecules, hybridize next to each other on the target DNA molecule. The first fluorescent dye, the donor dye fluorescein, is excited at 470 nm by the light source of the LightCycler. Instead of emitting light at 530 nm, the fluorescein can transfer its energy in a nonfluorescent manner to a reporter dye. The reporter dye emits light of a longer wavelength, e.g., 640 nm. This process, called fluorescence resonance energy transfer (FRET), enables real-time detection of the specific PCR product followed by melting curve analysis, which monitors the temperature-dependent hybridization with fluorescent oligonucleotide probes to single-stranded DNA. The success of this new technology in detecting single …