POINT MUTATION SCREENING FOR 16 EXONS OF THE DYSTROPHIN GENE BY MULTIPLEX SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS

We have developed a rapid and nonradioactive method to screen for poin t mutations using the Pharmacia PhastSystem. In an SSCP analysis, we a pplied the two multiplex exon PCR kits, commonly used for the detectio n of deletions in Duchenne and Becker muscular dystrophy patients. The different exon bands in the multiplex SSCP pattern could be identifie d by running well-characterised deletion patients in this system. Two common polymorphisms were easily identifiable and are helpful in the h aplotype analysis in families, Screening of 70 patients in which no gr oss rearrangement was detectable with the multiplex PCR and Southern b lot, resulted in the identification of 6 patients with a band shift af ter SSCP analysis. Of these 6 band shifts, 5 were the result of a fram e shift or termination mutation, The other band shift was found to be a rare polymorphism unlikely to be the cause of the patient's phenotyp e. Application of this technique enabled us to improve diagnosis in th e families involved and will allow us to extend the search for point m utations in the remaining exons of the dystrophin gene. (C) 1995 Wiley -Liss, Inc.