A Hemi-nested, Allele Specific, Whole Blood PCR Assay for the Detection of the Factor V Leiden Mutation

Gary D Sinclair; Sandra Low; Man-Chiu Poon

doi:10.1055/s-0038-1656129

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1997; 77(06): 1154-1155
DOI: 10.1055/s-0038-1656129

Coagulation

Schattauer GmbH Stuttgart

A Hemi-nested, Allele Specific, Whole Blood PCR Assay for the Detection of the Factor V Leiden Mutation

Gary D Sinclair

The Departments of Medical Biochemistry, Medicine, Paediatrics and Oncology, Faculty of Medicine, University of Calgary; Departments of Medicine and Laboratory Medicine, Foothills Hospital; and the Canadian Red Cross Society Blood Services, Calgary, Alberta, Canada

,

Sandra Low

The Departments of Medical Biochemistry, Medicine, Paediatrics and Oncology, Faculty of Medicine, University of Calgary; Departments of Medicine and Laboratory Medicine, Foothills Hospital; and the Canadian Red Cross Society Blood Services, Calgary, Alberta, Canada

,

Man-Chiu Poon

The Departments of Medical Biochemistry, Medicine, Paediatrics and Oncology, Faculty of Medicine, University of Calgary; Departments of Medicine and Laboratory Medicine, Foothills Hospital; and the Canadian Red Cross Society Blood Services, Calgary, Alberta, Canada

› Author Affiliations

Abstract

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Summary

We describe a novel hemi-nested, allele specific whole blood PCR assay for detection of the factor V Leiden mutation associated with the plasma defect, activated protein C resistance. This assay utilizes 5 μl of whole blood without prior DNA extraction. The hemi-nested design, employing an outer primer pair in combination with nested, allele specific primers obviates the need for restriction enzyme digestion. PCR reactions are analysed directly on agarose or polyacrylamide minigels. The assay confirmed the genotypes of 50 individuals previously categorized by PCR and Mnll digestion, and has been subsequently utilized in the genotyping of 445 individuals referred for thrombosis studies.

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References

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