Double fluorescent-amplification refractory mutation detection (dF-ARMS) of the factor V Leiden and prothrombin...

Simultaneous fluorescent [F] detection of the factor V Leiden (G1691A) and the prothrombin 3'-untranslated region (G20210A) mutations were performed in a single tube polymerase chain reaction (PCR). Amplification refractory mutation detection system (ARMS) formed the basis of this assay design. Fluorescent-labelled primers incorporated into amplicons during the reaction facilitated detection directly by GeneScan analysis without further manipulation. To test the efficacy of this double [F]-ARMS (dF-ARMS) method, 48 patients with unexplained thrombotic tendencies were investigated for their factor V Leiden and prothrombin genotypes. These results corresponded exactly with data achieved using the more conventional methods of restriction fragment length polymorphism (RFLP)-PCR and direct DNA sequencing. Three out of the 48 patients in this group were found to be compound heterozygotes.