Rapid Detection of Prothrombotic Mutations of Prothrombin (G20210A), FactorV (G1691A), and Methylenetetrahydrofolate Reductase (C677T) by Real-Time Fluorescence PCR with the LightCycler

Abstract

Analytical procedures for the identification of point mutations in genomic DNA are finding increasing application in the clinical laboratory. As the demand for these analyses grows, so does the need for rapid, reliable, and easy methods to detect known point mutations. Prothrombotic mutations can be found in almost 25% of unselected patients referred for thrombophilia workup (1). These include the factor V (G1691A), prothrombin (G20210A), and methylenetetrahydrofolate reductase (MTHFR; C677T) mutations (2)(3)(4). Recently, methods have been published for genotyping both the factor V (5) and the MTHFR (6) mutations by rapid cycle PCR using the LightCyclerTM (Roche Molecular Biochemicals). We describe here a procedure for genotyping the prothrombin mutation using the LightCycler. In addition, we have modified the PCR methods to perform all three PCR analyses in parallel, using the same program on the LightCycler. When this method is combined with a rapid DNA extraction, results can be obtained within 60 min after a whole blood sample is received. The appearance of a specific PCR product is monitored by adjacent hybridization probes (a labeled primer may also serve as a probe), that are usually designed to bind on one amplicon strand. The 3′ end of one probe is labeled with fluorescein (FLU), whereas the 5′ end of an adjacent probe is labeled with LC-Red640 (Roche Molecular Biochemicals) as the anchor probe. When both probes hybridize in close proximity, fluorescence resonance energy transfer (FRET) occurs, producing a specific fluorescence emission of LC-Red as a result of FLU excitation. Increasing the temperature during fluorescence reading yields a temperature/fluorescence curve from which the melting point of the probe can be derived. When the appropriate conditions are chosen, the mismatch under the detection probe caused by a single point mutation leads to a substantial decrease in the melting point …