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Clinical Chemistry 46: 1051-1056, 2000;
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(Clinical Chemistry. 2000;46:1051-1056.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Genotyping of Factor V G1691A (Leiden) without the Use of PCR by Invasive Cleavage of Oligonucleotide Probes

Martin J. Hessnera,1, Mary Ann Budish1 and Kenneth D. Friedman1

1 The Diagnostic Laboratories of The Blood Center of Southeastern Wisconsin, Milwaukee, WI 53201-2178.
a Address correspondence to this author at: The Blood Center, 638 North 18th St., PO Box 2178, Milwaukee, WI 53201-2178. Fax 414-937-6202; e-mail MJHessner{at}bcsew.edu

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 InvaderTM 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.




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