Genotyping of factor V G1691A (Leiden) without the use of PCR by invasive cleavage of oligonucleotide probes. Clin Chem 2000 Aug;46(8 Pt 1):1051-6
Date
08/06/2000Pubmed ID
10926882DOI
10.1093/clinchem/46.8.1051Scopus ID
2-s2.0-0033869814 (requires institutional sign-in at Scopus site) 34 CitationsAbstract
BACKGROUND: The factor V G1691A Leiden (FVL) mutation is the most common known hereditary risk factor for venous thrombosis.
METHODS: Third Wave Technologies, Inc. (Madison, WI) has developed a new microtiter plate-based assay that does not require PCR, restriction digestion, or gel electrophoresis. This technology system, termed the Invader(TM) assay, utilizes a 5' "invading" oligonucleotide and a partially overlapping 3' "signal" oligonucleotide, which together form a specific structure when bound to a complementary genomic DNA template. A thermostable flap endonuclease cleaves this structure, releasing the 5' flap from the signal oligonucleotide. Increased temperature and an excess of the signal probe enable multiple probes to be cleaved for each target sequence present without temperature cycling. The cleaved probes then direct cleavage of a secondary probe, which is 5' end-labeled with fluorescein but is quenched by an internal dye. Upon cleavage, the fluorescein-labeled product is detected using a standard fluorescence plate reader. Genotypes are determined by net wild-type/mutant signal ratio.
RESULTS: Complete concordance was observed, after resolution of four discordances, when 1369 individuals (1264 wild type, 102 heterozygous, 3 homozygous) were FVL genotyped by both the Invader assay and by allele-specific PCR.
CONCLUSION: We conclude that FVL genotyping using invasive cleavage of oligonucleotide probes is a rapid and reliable alternative to genotyping by more traditional PCR-based methods.
Author List
Hessner MJ, Budish MA, Friedman KDAuthors
Kenneth D. Friedman MD Professor in the Medicine department at Medical College of WisconsinMartin J. Hessner PhD Professor in the Pediatrics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
DNAFactor V
Fluorescence
Humans
Mutation
Nucleic Acid Hybridization
Oligonucleotide Probes
Polymerase Chain Reaction
