Improved detection of p53 mutations in soft tissue tumors using new gel composition for automated nonradioactive analysis of single-strand conformation polymorphism

• Published in: Electrophoresis Vol. 18; no. 15; p. 2849
• Main Authors: Schneider-Stock, R, Haeckel, C, Radig, K, Pein, C D, Roessner, A
• Format: Journal Article
• Language: English
• Published: Germany 1997
• Subjects: Autoanalysis; Exons; Fluorescence; Gels; Genes, p53; Genetic Testing - methods; Humans; Mutation; Polymorphism, Single-Stranded Conformational; Reproducibility of Results; Soft Tissue Neoplasms - genetics
• ISSN: 0173-0835
• DOI: 10.1002/elps.1150181521

We report a new nonradioactive method to detect sequence changes, including single-base substitutions through shifts in electrophoretic mobility using an automated fluorescence sequencer (ALFexpress, Pharmacia, Biotech) connected to external cooling equipment. Single strands were identified by incorporation of fluorescein-labeled primers during amplification and subsequent laser detection at the bottom of the gel. The amplified polymerase chain reaction (PCR) products were heat-denatured and loaded onto a polyacrylamide gel under nondenaturing conditions and strict control of constant low temperature. Peak shifts in the fluorogram indicated mutations. A novel gel composition improved the detection rate for mutations considerably. Automatic analysis of single-strand 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 soft tissue tumors. The mutation spectrum and frequency in exon 7 of the p53 gene are discussed with respect to oncogenesis in soft tissue sarcomas.