title: A microplate allele-specific oligonucleotide hybridization assay for detection of factor V Leiden.
abstract: Factor V Leiden is the most common genetic risk factor for thrombosis. Currently, the determination of factor V Leiden genotype is limited to laboratories with expertise in molecular methods to develop "home brew" assays using polymerase chain reaction (PCR) to amplify genomic DNA, followed by analysis of Mnl I restriction fragments. These methods are not standardized, are labor intensive, and have low throughput. We describe a method for determination of factor V Leiden genotype using allele-specific oligonucleotide capture probes coated onto 96 well plates, requiring only a thermal cycler and a microplate spectrophotometer to perform. With an automated strip washer and plate reader, genotypes could be determined in 80 min from completion of PCR. Within-run and between-run coefficients of variation for the assay were < 10%. In 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.
authors.affiliation: Molecular Diagnosis Laboratory, Stanford Health Services, Stanford University Medical Center, CA 94305, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Zehnder
authors.forename: R C
authors.initials: RC
authors.lastname: Benson
authors.forename: S
authors.initials: S
authors.lastname: Cheng
mesh_terms: D000483:Alleles; D005165:Factor V / Q000032:analysis / Q000235:genetics*; D005838:Genotype; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013924:Thrombophlebitis / Q000235:genetics
chemical_list: D015345:Oligonucleotide Probes; C095381:factor V Leiden; D005165:Factor V