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Clinical Chemistry 50: 296-305, 2004. First published December 4, 2003; 10.1373/clinchem.2003.023556
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Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 2004;50:296-305.)
© 2004 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

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

Cynthia French1, Conan Li1, Charles Strom1, Weimin Sun1, Reuel Van Atta2, Belen Gonzalez2 and Michael Wood2,a

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

aAddress correspondence to this author at: NAXCOR, 320 Logue Ave., Suite 200, Mountain View, CA 94043. Fax 650-903-9182; 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.




The following articles in journals at HighWire Press have cited this article:


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 Clin. Chem.Home page
G. Amicarelli, D. Adlerstein, E. Shehi, F. Wang, and G. M. Makrigiorgos
Genotype-Specific Signal Generation Based on Digestion of 3-Way DNA Junctions: Application to KRAS Variation Detection
Clin. Chem., October 1, 2006; 52(10): 1855 - 1863.
[Abstract] [Full Text] [PDF]


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