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Clinical Chemistry 45: 1898-1905, 1999;
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(Clinical Chemistry. 1999;45:1898-1905.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Detection of the Factor V Leiden Mutation by Direct Allele-specific Hybridization of PCR Amplicons to Photoimmobilized Locked Nucleic Acids

Henrik Ørum1,a, Mogens H. Jakobsen2, Troels Koch1, Jens Vuust3 and Martin B. Borre1

1 PNA Diagnostics A/S, Rønnegade 2, DK-2100 Copenhagen, Denmark.

2 Exiqon A/S, Bygstubben 9, DK-2950 Vedbæk, Denmark.

3 Department of Clinical Biochemistry, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark.
a Author for correspondence. Fax 45-44-44-03-73; e-mail oerum{at}euroconnect.dk

Background: Individuals carrying the factor V Leiden mutation have been shown to have an increased risk of developing venous thromboembolism. Our aim was to develop an ELISA-like assay to detect the mutation in PCR-amplified genomic DNA using novel, high-affinity DNA analogs, termed locked nucleic acids (LNAs).

Methods: LNA octamer probes complementary to the factor V wild-type or mutated sequence were covalently attached to individual wells of a microtiter plate. Biotinylated factor V amplicons were added, and hybridization to the immobilized LNA probes was scored colorimetrically using a horseradish peroxidase-anti-biotin Fab conjugate and tetramethylbenzidine substrate.

Results: In a prospective study of 53 patients, the assay reproducibly scored both factor V homozygotes and heterozygotes with excellent sensitivity and specificity. All results were in complete agreement with the results obtained with the conventional PCR-restriction fragment length polymorphism technique.

Conclusions: The simplicity of the assay and its procedural relatedness to the widely used ELISA format should make it useful for routine factor V testing in the clinical laboratory.




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