Allelic discrimination of factor V Leiden using a 5 ' nuclease assay

The G1691A (Leiden) mutation of the factor V gene is the most prevalent ide ntified cause of venous thrombosis. Therefore, we developed a new genetic t est using the TaqMan system. With this assay which combines PCR amplificati on and detection reaction in one closed tube, a cohort of 234 patients with a history of thrombosis was screened. In parallel, amplification products of the same patients were screened with a previously described test using e ndonuclease digestion of PCR products followed by gel electrophoresis, Iden tical results were obtained by both methods. Among cases, 122 (52%) individ uals were homozygous normal, 99 (42%) were heterozygous affected and 13 (5. 5%) showed homozygous pattern for the Factor V Leiden mutation, Thus, it co uld be demonstrated that the new TaqMan assay is a robust, rapid and automa ted method for high throughput application which avoids time consuming and difficult post-PCR steps.