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 humans tumors, but their clinical significance remains to be precisely elucidated. Conventional single-strand conformation polymorphism (SSCP) analysis, a well-established technique for detecting p53 mutations, uses radioactively 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 ovarian carcinomas and two cell lines (SW480 and Caov4) harboring known homozygous p53 mutations and compared the results with nonradioactive silver-stained SSCP. The purpose was to assess whether nonradioactive SSCP is suitable for detecting p53 mutations in a rapid, sensitive, cost-effective fashion, without the need of radioactive isotopes. We accomplished PCR amplification of p53 exons 5 through 8 in 26 carcinomas, and radioactive SSCP detected p53 mutations in 13 tumors; three mutations 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 SSCP, except for one exon 8 mutation. To establish the sensitivity of nonradioactive SSCP, DNA samples of SW480 and Caov4 were mixed with increasing amounts (0-90%) of normal DNA and subjected to PCR-SSCP analysis. 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 large series of patients to assess the clinical significance of p53 mutations in human tumors.
Bosari
S
S
Department of Pathology, University of Milan School of Medicine, Italy.
Marchetti
A
A
Buttitta
F
F
Graziani
D
D
Borsani
G
G
Loda
M
M
Bevilacqua
G
G
Coggi
G
G
D002277:Carcinoma / Q000235:genetics / Q000473:pathology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005091:Exons / Q000235:genetics; D005260:Female; D016158:Genes, p53 / Q000235:genetics*; D006801:Humans; D010051:Ovarian Neoplasms / Q000235:genetics / Q000473:pathology; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D011849:Radioactive Tracers; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016622:Silver Staining / Q000379:methods*; D014407:Tumor Cells, Cultured / Q000473:pathology
D011849:Radioactive Tracers