title: Development of a rapid DNA screening procedure for the Factor V Leiden mutation.
abstract: Aim-To develop a rapid, simple and highly specific DNA screening procedure based on the amplification refractory mutation system (ARMS) to detect the Leiden mutation in whole blood.Methods-ARMS PCR amplification primers with additional mismatches at either -2 or -3, which greatly improves specificity, were constructed to detect the normal Factor V gene and the Leiden mutation in whole blood samples from patients with abnormal clotting results.Results-Construction of ARMS primers with either an additional mismatch at -2 or -3 at the 3' end of the primer could be used to detect the Leiden mutation in 0.5 mu1 whole blood in under three hours. Primers destabilised at position -3 could be used at a lower annealing temperature, which gave greater sensitivity and are now routinely used. A control set of primers was included in the same reaction to act as a positive control.Conclusions-This rapid and specific assay for the factor V Leiden mutation is a useful addition to the investigation of patients with or at risk from thrombovascular disease.
authors.affiliation: Division of Clinical Laboratory Sciences, Department of Clinical Chemistry, Queens Medical Centre, Nottingham.
authors.forename: G A
authors.initials: GA
authors.lastname: Scobie
authors.forename: S T
authors.initials: ST
authors.lastname: Ho
authors.forename: G
authors.initials: G
authors.lastname: Dolan
authors.forename: N A
authors.initials: NA
authors.lastname: Kalsheker