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 mo lecular basis for resistance to activated protein C (APC-R). This muta tion has been reported in approximately 20 - 50% of individuals with p reviously unexplained thrombophilia and 3 - 5% of the general populati on. We have adapted an oligonucleotide ligation assay (OLA) for noniso topic detection of the FV:Q(506) mutation which permits rapid screenin g for this mutation. First, the polymerase chain reaction (PCR) was us ed for target DNA amplification, thus permitting nonisotopic reporters in the DNA analysis. Then thermostable ligase was used for ligation o r covalent coupling of adjacent wild-type and mutant oligonucleotide p robes which occurs only when the probes are annealed to a matched ampl icon. A colorimetric ELISA-based detection assay was then used to capt ure 5' biotinylated probes in 96-well streptavidin-coated plates and b y virtue of ligation, detection of a 3' digoxigenin reporter probe. Fo llowing the addition of anti-digoxigenin conjugate and enhanced alkali ne phosphatase signal amplification, colorimetric substrate change was measured in an ELISA plate reader. This assay correctly identified FV genotypes of 290 samples.