IMPROVED DETECTION OF P53 MUTATIONS IN SOFT-TISSUE TUMORS USING NEW GEL COMPOSITION FOR AUTOMATED NONRADIOACTIVE ANALYSIS OF SINGLE-STRAND CONFORMATION POLYMORPHISM

We report a new nonradioactive method to detect sequence changes, incl uding single-base substitutions through shifts in electrophoretic mobi lity using an automated fluorescence sequencer (ALFexpress, Pharmacia, Biotech) connected to external cooling equipment. Single strands were identified by incorporation of fluorescein-labeled primers during amp lification and subsequent laser detection at the bottom of the gel. Th e amplified polymerase chain reaction (PCR) products were heat-denatur ed and loaded onto a polyacrylamide gel under nondenaturing conditions and strict control of constant low temperature. Peak shifts in the fl uorogram indicated mutations. A novel gel composition improved the det ection rate for mutations considerably. Automatic analysis of single-s trand conformation polymorphism (SSCP) gels saves time and costs, and is highly reproducible. The method was applied for mutation screening in exon 7 of the p53 tumor suppressor gene in DNA of freshly frozen so ft tissue tumors. The mutation spectrum and frequency in exon 7 of the p53 gene are discussed with respect to oncogenesis in soft tissue sar comas.