A MICROPLATE ALLELE-SPECIFIC OLIGONUCLEOTIDE HYBRIDIZATION ASSAY FOR DETECTION OF FACTOR-V-LEIDEN

Factor V Leiden is the most common genetic risk factor for thrombosis. Currently, the determination of factor V Leiden genotype is limited t o laboratories with expertise in molecular methods to develop ''home b rew'' assays using polymerase chain reaction (PCR) to amplify genomic DNA, followed by analysis of Mnl I restriction fragments. These method s are not standardized, are labor intensive, and have low throughput. We describe a method for determination of factor V Leiden genotype usi ng allele-specific oligonucleotide capture probes coated onto 96 well plates, requiring only a thermal cycler and a microplate spectrophotom eter to perform. With an automated strip washer and plate reader, geno types could be determined in 80 min from completion of PCR. Within-run and between-run coefficients of variation for the assay were < 10%. I n all 160 cases studied, the microplate assay correctly identified the factor V genotype. The microplate oligonucleotide hybridization assay is a simple and reliable system for determination of factor V Leiden genotype. The assay offers an automatable, high-throughput alternative to current testing methodologies.