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, 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.
Schneider-Stock
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R
Department of Pathology, Otto-von-Guericke University, Magdeburg, Germany. Regine.Schneider-Stock@medizin.uni-magdeburg.de
Haeckel
C
C
Radig
K
K
Pein
C D
CD
Roessner
A
A
D001322:Autoanalysis; D005091:Exons; D005453:Fluorescence; D005782:Gels; D016158:Genes, p53; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D009154:Mutation; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results; D012983:Soft Tissue Neoplasms / Q000235:genetics
D005782:Gels