Oligonucleotide ligation assay for detection of the factor V mutation (arg(506)->Gln) causing protein C resistance

A point mutation in the Factor V (FV) gene at the activated protein C cleavage site, FV Arg (R)(506) --> FV GLn (Q)(506), is the reported molecular basis for resistance to activated protein C (APC-R). This mutation has been reported in approximately 20 - 50% of individuals with previously unexplained thrombophilia and 3 - 5% of the general population. We have adapted an oligonucleotide ligation assay (OLA) for nonisotopic detection of the FV:Q(506) mutation which permits rapid screening for this mutation. First, the polymerase chain reaction (PCR) was used for target DNA amplification, thus permitting nonisotopic reporters in the DNA analysis. Then thermostable ligase was used for ligation or covalent coupling of adjacent wild-type and mutant oligonucleotide probes which occurs only when the probes are annealed to a matched amplicon. A colorimetric ELISA-based detection assay was then used to capture 5' biotinylated probes in 96-well streptavidin-coated plates and by virtue of ligation, detection of a 3' digoxigenin reporter probe. Following the addition of anti-digoxigenin conjugate and enhanced alkaline phosphatase signal amplification, colorimetric substrate change was measured in an ELISA plate reader. This assay correctly identified FV genotypes of 290 samples.