Allelic discrimination of factor V Leiden using a 5' nuclease assay.
The G1691A (Leiden) mutation of the factor V gene is the most prevalent identified cause of venous thrombosis. Therefore, we developed a new genetic test using the TaqMan system. With this assay which combines PCR amplification 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 endonuclease digestion of PCR products followed by gel electrophoresis. Identical results were obtained by both methods. Among cases, 122 (52%) individuals were homozygous normal, 99 (42%) were heterozygous affected and 13 (5.5%) showed homozygous pattern for the Factor V Leiden mutation. Thus, it could be demonstrated that the new TaqMan assay is a robust, rapid and automated method for high throughput application which avoids time consuming and difficult post-PCR steps.
Happich
D
D
Institute of Experimental Haematology and Transfusion Medicine, University of Bonn, Germany.
Schwaab
R
R
Hanfland
P
P
Hoernschemeyer
D
D
D000483:Alleles; D001681:Biological Assay; D017931:DNA Primers; D004720:Endonucleases; D005165:Factor V / Q000235:genetics*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers; C095381:factor V Leiden; D005165:Factor V; D004720:Endonucleases