Abstract
p53 is a tumor suppressor gene located on 17p, a region of the human genome frequently deleted in tumors. Mutation of the p53 gene is an important step leading to development of many forms of human cancer. To simplify the analysis of tumors for p53 point mutations, we describe a GC-clamped denaturing gradient gel assay for detecting single-base substitutions within highly conserved regions of the p53 gene. This assay alows for efficient screening of tumors for single-base substitutions within the p53 gene and can be used to facilitate sequence analysis of p53 point mutations.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Børresen AL, Hovig E, Smith-Sorensen B, Malkin D, Lystad S, Andersen TI, Nesland JM, Isselbacher KJ, Friend SH (1991) Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations. Proc Natl Acad Sci USA 88:8405–8409
Bressac B, Kew M, Wanda J, Ozturk M (1991) Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature 350:429–431
Cogen PH, Daneshvar L, Metzger AK, Duyk G, Edwards MSB, Sheffield VC (1992) Involvement of multiple chromosome 17p loci in medulloblastoma tumorigenesis. Am J Hum Genet 50:584–589
Cotton RGH, Rodriquez NR, Campbell RD (1988) Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study of mutations. Proc Natl Acad Sci USA 85:4397–4401
Fenaux P, Jonveaux P, Quiquandon I, Lai JL, Pignon JM, Loucheux-Lefebvre MH, Bauters F, Berger R, Kerckaert JP (1991) p53 genemutations in acute myeloid leukemia with 17p monosomy. Blood 78:1652–1657
Fischer SG, Lerman LS (1980) Separation of random fragments of DNA according to properties of their sequences. Proc Natl Acad Sci USA 77:4420–4424
Gaidano G, Ballerini P, Gong JZ, Inghirami G, Neri A, Newcomb EW, Magrath IT, Knowles DM, Dalla-Favera R (1991) p53 mutations in human lymphoid malignancies: association with Burkitt lymphoma and chronic lymphocytic leukemia. Proc Natl Acad Sci USA 88:5413–5417
Hayashi K (1992) PCR-SSCP: a simple and sensitive method for detection of mutations in the genomic DNA. PCR Methods Appl 1:34–38
Hollstein M, Sidransky D, Vogelstein B, Harris CC (1991) p53 mutations in human cancers. Science 253:49–53
Hsu IC, Metcalf RA, Sun T, Welsh JA, Wang NJ, Harris CC (1991) Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature 350:427–428
Jonveaux PH, Fenaux P, Quiquandon I, Pignon JM, Lai JL, Loucheux-Lefebvre MH, Goossens M, Bauters F, Berger R (1991) Mutations in the p53 gene in myelodysplastic syndromes. Oncogene 6:2243–2247
Lerman LS, Silverstein K (1987) Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. Methods Enzymol 155:482–501
Levine AJ, Momand J, Finlay C (1991) The p53 tumour suppressor gene. Nature 351:453–456
Malkin D, Li FP, Strong LC, Fraumeni JF Jr, Nelson CE, Kim DH, Kassel J, Gryka MA, Bischoff FZ, Tainsky MA, Friend SH (1990) Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science 250:1233–1238
Metzger AK, Sheffield VC, Duyk G, Daneshvar L, Edwards MSB, Cogen PH (1991) Identification of a germ-line mutation in the p53 gene in a patient with an intracranial ependymoma. Proc Natl Acad Sci USA 88:7825–7829
Mulligan LM, Matlashewski GJ, Scrable HJ, Cavenee WK (1990) Mechanisms of p53 loss in human sarcomas. Proc Natl Acad Sci USA 87:5863–5867
Myers RM, Larin Z, Maniatis T (1985a) Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA: DNA duplexes. Science 230:1242–1246
Myers RM, Fisher SG, Lerman LS, Maniatis T (1985b) Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res 13:3131–3145
Myers RM, Maniatis T, Lerman LS (1987) Detection and localization of single base changes by denaturing gradient gel electrophoresis. Methods Enzymol 155:501–527
Nigro JM, Baker SJ, Preisinger AC, Jessup JM, Hotstetter R, Cleary K, Bigner SH et al (1989) Mutations in the p53 gene occur in diverse tumor types. Nature 342:705–708
Orita M, Iwahara H, Kanazawa H, Hayashi K, Sekiya T (1989a) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA 86:2766–2770
Orita MY, Suzuki Y, Sekiya T, Hayashi K (1989b) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874–879
Sheffield VC, Cox DR, Myers RM (1989) Attachment of a 40-base pair G + C rich sequence to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci USA 86:232–236
Sheffield VC, Fishman GA, Beck JS, Kimura AE, Stone EM (1991) Identification of novel rhodopsin mutations associated with retinitis pigmentosa by GC-clamped denaturing gradient gel electrophoresis. Am J Hum Genet 49:699–706
Sheffield VC, Beck JS, Nichols B, Cousineau A, Lidral A, Stone EM (1992) Detection of multiallele polymorphisms within gene sequences by GC-clamped denaturing gradient gel electrophoresis. Am J Hum Genet 50:567–575
Toguchida J, Yamaguchi T, Dayton SH, Beauchamp RL, Herrera GE, Ishikazi K, Yamamuro T, et al (1992) Prevalence and spectrum of germline mutations of the p53 gene among patients with sarcoma. N Engl J Med 326:1303–1308
Zhou Y, Zhang X, Ebright RH (1991) Random mutagenesis of genesized DNA molecules by use of PCR with Taq DNA polymerase. Nucleic Acids Res 19:6052
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Beck, J.S., Kwitek, A.E., Cogen, P.H. et al. A denaturing gradient gel electrophoresis assay for sensitive detection of p53 mutations. Hum Genet 91, 25–30 (1993). https://doi.org/10.1007/BF00230217
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00230217