Abstract
A protocol for detection of mutations in the TP53 gene using temporal temperature gradient 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 and constant denaturant gel electrophoresis, and eliminates some of the problems. The result is a rapid and sensitive screening technique which 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
References
Hernandez-Boussard T, Montesano R, 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
Olivier M, Eeles R, Hollstein M et al (2002) The IARC TP53 database: new online mutation analysis and recommendations to users. Hum Mutat 19:607–614
Olivier M, Hollstein M, Hainaut P (2010) TP53 mutations in human cancers: origins, consequences, and clinical use. Cold Spring Harb Perspect Biol 2:a001008
Hollstein M, Rice K, Greenblatt MS, Soussi T, Fuchs R, Sørlie T, Hovig E, Smith-Sorensen B, Montesano R, Harris CC (1994) Database of p53 gene somatic mutations in human tumors and cell lines. Nucleic Acids Res 22:3551–3555
Hussain SP, Hofseth LJ, Harris CC (2001) Tumor suppressor genes: at the crossroads of molecular carcinogenesis, molecular epidemiology and human risk assessment. Lung Cancer 34(suppl 2):S7–S15
Martin AC, Facchiano AM, Cuff AL et al (2002) Integrating mutation data and structural analysis of the TP53 tumor-suppressor protein. Hum Mutat 19:149–164
Soussi T, Beroud C (2002) Assessing TP53 status in human tumours to evaluate clinical outcome. Nat Rev Cancer 1:233–240
Tyner SD, 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 RA, Meling GI, Hainaut P, Rognum TO, 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 PE, Aas T et al (2001) Influence of TP53 gene alterations and c-erbB2 expression on the response to treatment with doxorubicin in locally advanced breast cancer. Cancer Res 61:2505–2512
Wallace-Brodeur RR, Lowe SW (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, Sekiya T (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci U S A 86:2766–2770
Orita M, Suzuki Y, Sekiya T, Hayashi K (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874–879
Fischer SG, Lerman LS (1983) DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc Natl Acad Sci U S A 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 U S A 88: 8405–8409
Hovig E, Smith-Sorensen B, Brogger A, 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, Ekstrom PO (2001) Mutation analysis of TP53 exons 5–8 by automated constant denaturant capillary electrophoresis. Tumour Biol 22:323–327
Khrapko K, Hanekamp JS, Thilly WG, Belenkii A, Foret F, Karger BL (1994) Constant denaturant capillary electrophoresis (CDCE): a high resolution approach to mutational analysis. Nucleic Acids Res 22:364–369
Sarkar G, Yoon HS, Sommer SS (1992) Dideoxy fingerprinting (ddE): a rapid and efficient screen for the presence of mutations. Genomics 13:441–443
Gelfi C, Cremonesi L, Ferrari M, Righetti PG (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, Langeroed A (1997) Temporal temperature gradient electrophoresis on the DCode system. Biorad Bull 2133:8
Zoller P, Redila-Flores T, Chu D, Patel A (1998) Temporal temperature gradient electrophoresis: a powerful technique to screen mutations. Biomedical Products 9. (http://www.biocompare.com/Application-Notes/42665-Temporal-Temperature-Gradient-Electrophoresis-A-Powerful-Technique-To-Screen-Mutations/)
Lerman LS, 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: Pfeifer GP (ed) Technologies for detection of DNA damage and mutation. Plenum, New York, pp 267–279
Kraggerud SM, Szymanska J, Abeler VM et al (2000) DNA copy number changes in malignant ovarian germ cell tumors. Cancer Res 60: 3025–3030
Sheffield VC, Cox DR, Lerman LS, Myers RM (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 U S A 86: 232–236
Guldberg P, Nedergaard T, Nielsen HJ, Olsen AC, Ahrenkiel V, 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
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Humana Press
About this protocol
Cite this protocol
Sørlie, T., Johnsen, H., Vu, P., Lind, G.E., Lothe, R., Børresen-Dale, AL. (2014). Mutation Screening of the TP53 Gene by Temporal Temperature Gel Electrophoresis (TTGE). In: Keohavong, P., Grant, S. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology, vol 1105. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-739-6_25
Download citation
DOI: https://doi.org/10.1007/978-1-62703-739-6_25
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-738-9
Online ISBN: 978-1-62703-739-6
eBook Packages: Springer Protocols