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Oligonucleotide ligation assay for detection of the factor V mutation (Arg506-->Gln) causing protein C resistance
J M Benson1, D J Phillips, B P Holloway
1Hematologic Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
Abstract:
A point mutation in the Factor V (FV) gene at the activated protein C cleavage site, FV Arg (R)506-->FV Gln (Q)506, is the reported molecular basis for resistance to activated protein C (APC-R). This mutation has been reported in approximately 20-50% of individuals with previously unexplained thrombophilia and 3-5% of the general population. We have adapted an oligonucleotide ligation assay (OLA) for nonisotopic detection of the FV:Q506 mutation which permits rapid screening for this mutation. First, the polymerase chain reaction (PCR) was used for target DNA amplification, thus permitting nonisotopic reporters in the DNA analysis. Then thermostable ligase was used for ligation or covalent coupling of adjacent wild-type and mutant oligonucleotide probes which occurs only when the probes are annealed to a matched amplicon. A colorimetric ELISA-based detection assay was then used to capture 5' biotinylated probes in 96-well streptavidin-coated plates and by virtue of ligation, detection of a 3' digoxigenin reporter probe. Following the addition of anti-digoxigenin conjugate and enhanced alkaline phosphatase signal amplification, colorimetric substrate change was measured in an ELISA plate reader. This assay correctly identified FV genotypes of 290 samples.
Insights
A new assay rapidly screens for the Factor V (FV) Q506 mutation, a common cause of thrombophilia. This nonisotopic oligonucleotide ligation assay accurately identifies genetic variations linked to blood clotting disorders.
Area of Science:
- Genetics
- Molecular Biology
- Hematology
Background:
- Activated protein C resistance (APC-R) is often caused by a specific Factor V (FV) gene mutation (FV Q506).
- This FV Q506 mutation is prevalent in individuals with unexplained thrombophilia and the general population.
- Accurate and rapid genetic screening for the FV Q506 mutation is crucial for diagnosing thrombophilia.
Purpose of the Study:
- To adapt and validate a nonisotopic oligonucleotide ligation assay (OLA) for rapid detection of the FV Q506 mutation.
- To enable efficient genetic screening for activated protein C resistance (APC-R).
Main Methods:
- Utilized polymerase chain reaction (PCR) for target DNA amplification.
- Employed a thermostable ligase for specific ligation of wild-type and mutant oligonucleotide probes to amplicons.
- Developed a colorimetric ELISA-based detection system using biotin and digoxigenin reporters for signal amplification and measurement.
Main Results:
- The developed OLA assay demonstrated accurate identification of Factor V (FV) genotypes.
- The assay successfully genotyped 290 samples, confirming its reliability.
- The nonisotopic detection method allows for rapid screening of the FV Q506 mutation.
Conclusions:
- The adapted oligonucleotide ligation assay provides a rapid, nonisotopic, and accurate method for detecting the FV Q506 mutation.
- This assay is effective for screening populations for genetic predispositions to thrombophilia.
- The OLA method offers a valuable tool for clinical diagnostics and genetic research in hematology.