title: Cross-linking hybridization assay for direct detection of factor V Leiden mutation.
abstract: A nucleic acid photocross-linking technology was used in the development of a direct assay for factor V Leiden, a point mutation in the factor V gene (G1691A) that is the most common inherited risk factor for thrombosis. This cross-linking hybridization assay included two allele-specific capture probes and six signal-generating reporter probes; all were modified with a photoactivated cross-linking compound. By using two different capture probes complementary to a 16-base sequence at the factor V Leiden mutation site, but differing in the nucleotide opposite the mutation site (C vs T), wild-type and factor V Leiden alleles were differentiated in purified DNA specimens. The assay was also successfully applied to genomic DNA in leukocytes isolated from whole blood; the factor V status of 122 patients as determined by this method was in complete concordance with a standard PCR-based assay and clearly discriminated between healthy individuals and factor V Leiden heterozygotes.
authors.affiliation: Molecular Diagnosis Laboratory, Stanford Health Services, Stanford University Medical Center, CA 94305, USA. zehnder@leland.stanford.edu
authors.forename: J
authors.initials: J
authors.lastname: Zehnder
authors.forename: R
authors.initials: R
authors.lastname: Van Atta
authors.forename: C
authors.initials: C
authors.lastname: Jones
authors.forename: H
authors.initials: H
authors.lastname: Sussman
authors.forename: M
authors.initials: M
authors.lastname: Wood
mesh_terms: D000483:Alleles; D001483:Base Sequence; D003432:Cross-Linking Reagents; D004912:Erythrocytes / Q000737:chemistry; D005091:Exons; D005165:Factor V / Q000032:analysis* / Q000235:genetics; D005838:Genotype; D006801:Humans; D009154:Mutation; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012307:Risk Factors; D013927:Thrombosis / Q000453:epidemiology / Q000235:genetics
chemical_list: D003432:Cross-Linking Reagents; D015345:Oligonucleotide Probes; C095381:factor V Leiden; D005165:Factor V