Fluorescence-based SSCP Analysis with Automatic Allele Detection Demonstrated for the Factor V Leiden Mutation

Abstract

Resistance of coagulation factor V to activated protein C is caused by a point mutation in which a G→A substitution at nucleotide 1691 of the factor V gene leads to the replacement of Arg506 by Gln (1) . Because of the high prevalence of the factor V Leiden mutation, it would be reasonable to perform a rapid and simple DNA test. In recent publications, several strategies of DNA testing were described. These tests included restriction enzyme digestion, the introduction of new cutting sites, allele-specific PCR, oligonucleotide ligation assay, and single-strand conformation polymorphism (SSCP) analysis (1)(2)(3)(4)(5) . The high sensitivity in mutation detection and the simple application have led to wide use of the SSCP method. Currently in most laboratories, SSCP analysis is performed in polyacrylamide gels in combination with silver staining. In this report, we adapted SSCP analysis to capillary electrophoresis. This was the first step toward automation because preparation of a gel was no longer necessary. The denatured DNA samples were placed in a sample tray, and after each electrophoretic run, new gel was injected from the system into the capillary. The SSCP patterns …