THE POLYMERASE CHAIN-REACTION WITH SEQUENCE-SPECIFIC PRIMERS FOR THE DETECTION OF THE FACTOR-V MUTATION ASSOCIATED WITH ACTIVATED PROTEIN-CRESISTANCE

The prevalence of the Factor V (FV) mutation associated with activated protein C resistance (FV Leiden) and its significance as a genetic ri sk factor for venous thrombosis have necessitated the development of a simple, rapid, and accurate assay for its detection. The polymerase c hain reaction with sequence specific primers (PCR-SSP) provides a powe rful technique for the discrimination of alleles resulting from single base substitutions. PCR amplification was performed using a sense pri mer complementary to both FV alleles coupled with either of two antise nse allele specific primers, one complementary to the normal FV allele and one complementary to the FV Leiden allele. PCR conditions were de veloped that favored amplification only in the case of perfect complem entation between template DNA and allele specific primer. The FV genot ype was assigned based on whether or not each allele specific primer s et produced an amplified product. Assignment of genotypes correlated 1 00% with those determined by the method of PCR amplification followed by Mn1I digestion. PCR-SSP allows the rapid and accurate identificatio n of carriers of the Factor V Leiden mutation by a simple PCR reaction without the need for the usual post-amplification specificity step.