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Thromb Haemost 1995; 74(03): 874-878
DOI: 10.1055/s-0038-1649840
Original Article
Coagulation
Schattauer GmbH Stuttgart

The Polymerase Chain Reaction with Sequence Specific Primers for the Detection of the Factor V Mutation Associated with Activated Protein C Resistance

Nancy E Kirschbaum
1   The Hemostasis Reference Laboratory of the Blood Center of Southeastern Wisconsin, USA
,
Paul A Foster
1   The Hemostasis Reference Laboratory of the Blood Center of Southeastern Wisconsin, USA
2   The Blood Research Institute of the Blood Center of Southeastern Wisconsin, USA
3   The Departments of Medicine, Pathology and Pediatrics of the Medical College of Wisconsin, Milwaukee, Wisconsin,USA
› Author Affiliations
Further Information

Publication History

Received 21 March 1995

Accepted after revision 23 May 1995

Publication Date:
09 July 2018 (online)

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Summary

The prevalence of the Factor V (FV) mutation associated with activated protein C resistance (FV Leiden) and its significance as a genetic risk factor for venous thrombosis have necessitated the development of a simple, rapid, and accurate assay for its detection. The polymerase chain reaction with sequence specific primers (PCR-SSP) provides a powerful technique for the discrimination of alleles resulting from single base substitutions. PCR amplification was performed using a sense primer complementary to both FV alleles coupled with either of two antisense allele specific primers, one complementary to the normal FV allele and one complementary to the FV Leiden allele. PCR conditions were developed that favored amplification only in the case of perfect complementation between template DNA and allele specific primer. The FV genotype was assigned based on whether or not each allele specific primer set produced an amplified product. Assignment of genotypes correlated 100% with those determined by the method of PCR amplification followed by MnII digestion. PCR-SSP allows the rapid and accurate identification of carriers of the Factor V Leiden mutation by a simple PCR reaction without the need for the usual post-amplification specificity step.

 

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