Development of standard reference materials for diagnosis of p53 mutations: analysis by slab gel single strand conformation polymorphism

Abstract

We have amplified by polymerase chain reaction (PCR) a 2.0 kbp region of the p53 gene containing exons 5--9 from seven cell lines reported in the literature to contain the majority of mutations reported for this gene. Sequence analysis of these products show that all seven cell lines contain mutations within the mutational hot spots of the p53 gene. Six of the seven clones have single base substitutions and the seventh has a single base deletion. We have analyzed the seven p53 single point mutations by single strand conformation polymorphism (SSCP) analysis using fluorescence slab gel electrophoresis (SG-SSCP). Fluorescent-labeled PCR primers were used for amplification of specific exons for mutation detection. SG-SSCP was conducted using Model 373 and Model 377 DNA sequencers with GeneScan Software (Perkin Elmer, Applied Biosystem Division). Nine different gel systems were first tested for their ability to resolve the p53 mutations using the Model 373 instrument. Two gel systems were capable of resolving all of the mutations that were screened. Optimal results were obtained with 12% w/v acrylamide 50:1 plus 10% v/v glycerol. This gel system was used to evaluate the effect of temperature on the ability to resolve the mutations. The separation with respect to wild type varied for each mutation examined. Subambient temperature (20 degrees C) was preferable overall for discrimination of these mutations as a group. We intend to use this system to examine a much larger panel of p53 mutation standards that are now under development.