Abstract
A protocol for detection of mutations in the TP53 gene using temporal temperature gradient gel electrophoresis (TTGE) is described. TTGE is a mutation detection technique that separates DNA fragments differing by single base pairs according to their melting properties in a denaturing gel. It is based on constant denaturing conditions in the gel combined with a temperature gradient during the electrophoretic run. This method combines some of the advantages of the related techniques denaturing gradient gel electrophoresis (DGGE) and constant denaturant gel electrophoresis (CDGE) and eliminates some of the problems. The result is a rapid and sensitive screening technique that is robust and easily set up in smaller laboratory environments.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Hernandez-Boussard, T., Montesano, R., and Hainaut, P. (1999) Sources of bias in the detection and reporting of p53 mutations in human cancer: analysis of the IARC p53 mutation database. Genet. Anal. 14, 229–233.
Hollstein, M., Rice, K., Greenblatt, M. S., et al. (1994) Database of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res. 22, 3551–3555.
Hussain, S. P., Hofseth, L. J., and Harris, C. C. (2001) Tumor suppressor genes: at the crossroads of molecular carcinogenesis, molecular epidemiology and human risk assessmen. Lung Cancer 34(suppl. 2), S7–S15.
Martin, A. C, Facchiano, A. M., Cuff, A. L., et al. (2002) Integrating mutation data and structural analysis of the TP53 tumor-suppressor protein. Hum. Mutat. 19, 149–164.
Soussi, T. and Beroud, C. (2002) Assessing TP53 status in human tumours to evaluate clinical outcome. Nat. Rev. Cancer 1, 233–240.
Tyner, S. D., Venkatachalam, S., Choi, J., et al. (2002) p53 mutant mice that display early ageing-associated phenotypes. Nature 415, 45–53.
Aas, T., Børresen, A.-L., Geisler, S., et al. (1996) Specific p53 mutations are associated with de novo resistance to doxorubicin in breast cancer patients. Nat. Med. 2, 811–814.
Børresen-Dale, A.-L., Lothe, R. A., Meling, G. I., Hainaut, P., Rognum, T. O., and Skovlund, E. (1998) TP53 and long-term prognosis in colorectal cancer: mutations in the L3 zinc-binding domain predict poor survival. Clin. Cancer Res. 4, 203–210.
Geisler, S., Lønning, P. E., Aas, T., et al. (2001) Influence of TP53 gene alterations and cerbB2 expression on the response to treatment with doxorubicin in locally advanced breast cancer. Cancer Res. 61, 2505–2512.
Wallace-Brodeur, R. R. and Lowe, S. W. (1999) Clinical implications of p53 mutations. Cell. Mol. Life Sci. 55, 64–75.
Wattel, E., Preudhomme, C., Hecquet, B., et al. (1994) p53 mutations are associated with resistance to chemotherapy and short survival in hematologic malignancies. Blood 84, 3148–3157.
Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K., and Sekiya, T. (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. USA 86, 2766–2770.
Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874–879.
Fischer, S. G. and Lerman, L. S. (1983) DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc. Natl. Acad. Sci. USA 80, 1579–1583.
Børresen, A.-L., Hovig, E., Smith-Sorensen, B., et al. (1991) Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations. Proc. Natl. Acad. Sci. USA 88, 8405–8409.
Hovig, E., Smith-Sorensen, B., Brogger, A., and Børresen, A.-L. (1991) Constant denaturant gel electrophoresis, a modification of denaturing gradient gel electrophoresis, in mutation detection. Mutat. Res. 262, 63–71 [Published erratum: Mutat. Res. 263, 61].
Bjorheim, J., Gaudernack, G., and Ekstrom, P. O. (2001) Mutation analysis of TP53 exons 5–8 by automated constant denaturant capillary electrophoresis. Tumour Biol. 22, 323–327.
Khrapko, K., Hanekamp, J. S., Thilly, W. G., Belenkii, A., Foret, F., and Karger, B. L. (1994) Constant denaturant capillary electrophoresis (CDCE): a high resolution approach to mutational analysis. Nucleic Acids Res. 22, 364–369.
Sarkar, G., Yoon, H. S., and Sommer, S. S. (1992) Dideoxy fingerprinting (ddF): a rapid and efficient screen for the presence of mutations. Genomics 13, 441–443.
Gelfi, C., Cremonesi, L., Ferrari, M., and Righetti, P. G. (1996) Temperature-programmed capillary electrophoresis for detection of DNA point mutations. BioTechniques 21, 926–928, 930, 932.
Riesner, D., Steger, G., Zimmat, R., et al. (1989). Temperature-gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein-nucleic acid interactions. Electrophoresis 10, 377–389.
Børresen-Dale, A.-L., Lystad, S., and Langeroed, A. (1997) Temporal temperature gradient electrophoresis on the DCode system. Biorad Bull. 2133.
Zoller, P., Redila-Flores, T., Chu, D., and Patel, A. (1998) Temporal temperature gradient electrophoresis—a powerful mutation screening technique. Biomed. Prod. 9.
Lerman, L. S. and Silverstein, K. (1987) Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. Methods Enzymol. 155, 482–501.
Børresen, A.-L. (1996) Constant denaturant gel electrophoresis (CDGE) in mutation screening, in Technologies for Detection of DNA Damage and Mutation (Pfeifer, G. P., ed.), Plenum, New York, pp. 267–279.
Kraggerud, S. M., Szymanska, J., Abeler, V. M., et al. (2000) DNA copy number changes in malignant ovarian germ cell tumors. Cancer Res. 60, 3025–3030.
Sheffield, V. C., Cox, D. R., Lerman, L. S., and Myers, R. M. (1989) Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) 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.
Guldberg, P., Nedergaard, T., Nielsen, H. J., Olsen, A. C, Ahrenkiel, V., and Zeuthen, J. (1997) Single-step DGGE-based mutation scanning of the p53 gene: application to genetic diagnosis of colorectal cancer. Hum. Mutat. 9, 348–355.
Steger, G. (1994) Thermal denaturation of double-stranded nucleic acids: prediction of temperatures critical for gradient gel electrophoresis and polymerase chain reaction. Nucleic Acids Res. 22, 2760–2768.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
Sørlie, T., Johnsen, H., Vu, P., Lind, G.E., Lothe, R., Børresen-Dale, AL. (2005). Mutation Screening of the TP53 Gene by Temporal Temperature Gradient Gel Electrophoresis. In: Keohavong, P., Grant, S.G. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology™, vol 291. Humana Press. https://doi.org/10.1385/1-59259-840-4:207
Download citation
DOI: https://doi.org/10.1385/1-59259-840-4:207
Publisher Name: Humana Press
Print ISBN: 978-1-58829-084-7
Online ISBN: 978-1-59259-840-3
eBook Packages: Springer Protocols