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Clinical Chemistry 0: 200302355, 2003; 10.1373/clinchem.2003.023556
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Received on June 23, 2003
Accepted on November 11, 2003

Molecular Diagnostics and Genetics

Detection of the Factor V Leiden Mutation by a Modified Photo-Cross-Linking Oligonucleotide Hybridization Assay

Cynthia French 1, Conan Li 1, Charles Strom 1, Weimin Sun 1, Reuel Van Atta 2, Belen Gonzalez 2, Michael Wood *

1 Quest Diagnostics Nichols Institute, San Juan Capistrano, CA
2 NAXCOR, Menlo Park, CA

* To whom correspondence should be addressed. E-mail: mikew{at}naxcor.com.

Background: Our previously developed assay for detection of the factor V Leiden mutation (G1691A) based on a nucleic acid photo-cross-linking technology used two allele-specific capture probes and six fluorescein-modified signal-generating reporter probes. We wished to improve the sensitivity and performance of the method.

Methods: We developed new reporter probes with ~10-fold more fluorescein molecules than the original probes. The single, cross-linker-modified capture probe was replaced by a three-probe system, separating the probe-target cross-linking function and the allelic differentiation function. The capture probe cross-linked to either or both of two flanking probes through stem structures at the capture-probe/flanking-probe junctions. The flanking probes cross-linked to target DNA through two cross-linking sites each. Genomic DNA was extracted from 0.2 mL of whole blood, and restriction-enzyme digested to create a defined 677 bp target sequence. Preliminary genotype ranges were determined for the assay by testing of pretyped samples. We then tested 1054 clinical samples, using an automated sample processor.

Results: The new assay had a 10-fold increase in signal-to-background ratio. Genotype results for 1039 of 1054 clinical samples (98.6%) agreed with those of a PCR-based method. Of the 15 remaining samples, 10 produced an indeterminate result outside the defined genotype ranges, 2 yielded insufficient signal to be genotyped, and 3 gave a discordant result. All 15 samples were genotyped correctly after reextraction of genomic DNA and retesting.

Conclusion: The modified photo-cross-linking assay for factor V Leiden detection is a sensitive non-PCR-based assay with potential for use in high-throughput clinical laboratories.© 2004 American Association for Clinical Chemistry


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[Abstract] [Full Text] [PDF]


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