A COLORIMETRIC MINISEQUENCING ASSAY FOR THE MUTATION IN CODON-506 OF THE COAGULATION-FACTOR-V GENE

We describe a colorimetric screening method for the mutation in codon 506 of the coagulation factor V gene. The nucleotide at the site of th e FV:Q(506) mutation is identified in an immobilized amplified DNA tem plate by extension of a primer with a hapten-labelled dNTP using a DNA polymerase. The incorporated hapten is detected by an antibody-alkali ne phosphatase conjugate that catalyses the formation of a coloured en d product. The assay is carried out in a microtiter plate format, and the procedure is identical to that of enzyme immunoassays. It unequivo cally identifies the FV:Q(506) mutation in heterozygous and homozygous form. The colorimetric minisequencing method gave the same result as a H-3-based minisequencing assay and restriction site analysis with Mn 11 used as reference methods. Because of its simple format and numeric result, the novel colorimetric minisequencing method should be an att ractive alternative for screening for the FV:Q(506) mutation in clinic al laboratories.