Detection of the factor V Leiden mutation by a modified photo-cross-linking oligonucleotide hybridization assay.
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 approximately 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 pre-typed 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 re-extraction of genomic DNA and retesting.


CONCLUSIONS
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.
French
Cynthia
C
Quest Diagnostics Nichols Institute, San Juan Capistrano, CA, USA.
Li
Conan
C
Strom
Charles
C
Sun
Weimin
W
Van Atta
Reuel
R
Gonzalez
Belen
B
Wood
Michael
M
D001322:Autoanalysis; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000528:radiation effects; D004247:DNA / Q000097:blood / Q000737:chemistry / Q000528:radiation effects; D015342:DNA Probes / Q000138:chemical synthesis / Q000528:radiation effects; D005165:Factor V / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry; D005838:Genotype; D006801:Humans; D009154:Mutation; D009693:Nucleic Acid Hybridization / Q000379:methods; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000528:radiation effects; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D015342:DNA Probes; D005456:Fluorescent Dyes; D009841:Oligonucleotides; C095381:factor V Leiden; D005165:Factor V; D004247:DNA