DETECTION OF P53 MUTATIONS BY SINGLE-STRAND CONFORMATION POLYMORPHISMS (SSCP) GEL-ELECTROPHORESIS - A COMPARATIVE-STUDY OF RADIOACTIVE AND NONRADIOACTIVE SILVER-STAINED SSCP ANALYSIS

p53 mutations are the most common genetic abnormality in human tumors, but their clinical significance remains to be precisely elucidated. C onventional single-strand conformation polymorphism (SSCP) analysis, a well-established technique for detecting p53 mutations, uses radioact ively labeled polymerase chain reaction (PCR) products, which migrate abnormally in the presence of mutations. We performed radioactive PCR- SSCP analysis in a series of 30 formalin-fixed, paraffin-embedded ovar ian carcinomas and two cell lines (SW480 and Caov4) harboring known ho mozygous p53 mutations and compared the results with nonradioactive si lver-stained SSCP. The purpose was to assess whether nonradioactive SS CP is suitable for detecting p53 mutations in a rapid, sensitive, cost -effective fashion, without the need of radioactive isotopes. We accom plished PCR amplification of p53 exons 5 through 8 in 26 carcinomas, a nd radioactive SSCP detected p53 mutations in 13 tumors: three mutatio ns were localized in exon 5, six in exon 6, two in exon 7, and two in exon 8. All mutations were correctly identified with nonradioactive SS CP, except for one exon 8 mutation. To establish the sensitivity of no nradioactive SSCP, DNA samples of SW480 and Caov4 were mixed with incr easing amounts (0-90%) of normal DNA and subjected to PCR-SSCP analysi s. Mutations were detected until the concentration of SW480 and Caov4 was 15% and 10%, respectively, of the total sample, The results of our investigation demonstrate that nonradioactive silver-stained SSCP is a sensitive, rapid, and Simple technique to detect p53 mutations, even in formalin-fixed tissues, and could be easily used to investigate la rge series of patients to assess the clinical significance of p53 muta tions in human tumors.