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

Electrophoresis. 1997 Dec;18(15):2849-51. doi: 10.1002/elps.1150181521.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autoanalysis
  • Exons
  • Fluorescence
  • Gels*
  • Genes, p53*
  • Genetic Testing / methods*
  • Humans
  • Mutation
  • Polymorphism, Single-Stranded Conformational*
  • Reproducibility of Results
  • Soft Tissue Neoplasms / genetics

Substances

  • Gels