Rapid analysis of factor V Leiden mutation by membrane mediated restriction digestion and ultra-thin layer gel electrophoresis

Mutation of blood coagulation factor V at the protein C cleavage site (Leiden mutation) results in activated protein C resistancy and concomitant blood clotting. Individuals that carry this mutation have significantly increased risk of thrombic episodes. Using polymerase chain reaction/ restriction fragment length polymorphism (PCR/RFLP) analysis, single nucleotide polymorphisms (SNP) such as the factor V Leiden mutation can be readily detected. In this paper, we discuss the applicability of membrane mediated restriction endonuclease digestion in sub-microliter volumes, in conjunction with ultra-thin layer agarose gel electrophoresis for rapid genotyping of factor V Leiden. The amplified and restriction enzyme digested DNA fragments were fluorescently labeled with ethidium bromide during the electrophoresis separation process. Combining membrane mediated restriction digestion with ultra-thin layer agarose gel electrophoresis separation and sensitive on-line laser-induced fluorescence detection enabled completion both the digestion and electrophoresis analysis procedures in less than 15 minutes for up to 96 samples, thus, genotyping of factor V Leiden mutation with this new protocol can be accomplished in a sensitive and high throughput manner.