Detection of the factor V Leiden mutation by direct allele-specific hybridization of PCR amplicons to photoimmobilized locked nucleic acids.
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.
Orum
H
H
PNA Diagnostics A/S, Ronnegade 2, DK-2100 Copenhagen, Denmark. oerum@euroconnect.dk
Jakobsen
M H
MH
Koch
T
T
Vuust
J
J
Borre
M B
MB
D000483:Alleles; D000880:Anthraquinones; D004797:Enzyme-Linked Immunosorbent Assay; D005165:Factor V / Q000235:genetics*; D006801:Humans; D009154:Mutation; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction / Q000379:methods; D011446:Prospective Studies; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014466:Ultraviolet Rays
D000880:Anthraquinones; D015345:Oligonucleotide Probes; C095381:factor V Leiden; D005165:Factor V