Mutation Screening of the TP53 Gene by Temporal Temperature Gradient Gel Electrophoresis

  • Therese Sørlie
  • Hilde Johnsen
  • Phuong Vu
  • Guro Elisabeth Lind
  • Ragnhild Lothe
  • Anne-Lise Børresen-Dale
Part of the Methods in Molecular Biology™ book series (MIMB, volume 291)

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.

Key Words

TP53 TTGE DGGE CDGE mutation screening 

References

  1. 1.
    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.PubMedGoogle Scholar
  2. 2.
    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.PubMedGoogle Scholar
  3. 3.
    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.PubMedCrossRefGoogle Scholar
  4. 4.
    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.PubMedCrossRefGoogle Scholar
  5. 5.
    Soussi, T. and Beroud, C. (2002) Assessing TP53 status in human tumours to evaluate clinical outcome. Nat. Rev. Cancer 1, 233–240.CrossRefGoogle Scholar
  6. 6.
    Tyner, S. D., Venkatachalam, S., Choi, J., et al. (2002) p53 mutant mice that display early ageing-associated phenotypes. Nature 415, 45–53.PubMedCrossRefGoogle Scholar
  7. 7.
    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.PubMedCrossRefGoogle Scholar
  8. 8.
    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.PubMedGoogle Scholar
  9. 9.
    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.PubMedGoogle Scholar
  10. 10.
    Wallace-Brodeur, R. R. and Lowe, S. W. (1999) Clinical implications of p53 mutations. Cell. Mol. Life Sci. 55, 64–75.PubMedCrossRefGoogle Scholar
  11. 11.
    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.PubMedGoogle Scholar
  12. 12.
    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.PubMedCrossRefGoogle Scholar
  13. 13.
    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.PubMedCrossRefGoogle Scholar
  14. 14.
    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.PubMedCrossRefGoogle Scholar
  15. 15.
    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.PubMedCrossRefGoogle Scholar
  16. 16.
    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].PubMedCrossRefGoogle Scholar
  17. 17.
    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.PubMedCrossRefGoogle Scholar
  18. 18.
    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.PubMedCrossRefGoogle Scholar
  19. 19.
    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.PubMedCrossRefGoogle Scholar
  20. 20.
    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.PubMedGoogle Scholar
  21. 21.
    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.PubMedCrossRefGoogle Scholar
  22. 22.
    Børresen-Dale, A.-L., Lystad, S., and Langeroed, A. (1997) Temporal temperature gradient electrophoresis on the DCode system. Biorad Bull. 2133.Google Scholar
  23. 23.
    Zoller, P., Redila-Flores, T., Chu, D., and Patel, A. (1998) Temporal temperature gradient electrophoresis—a powerful mutation screening technique. Biomed. Prod. 9.Google Scholar
  24. 24.
    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.PubMedCrossRefGoogle Scholar
  25. 25.
    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.Google Scholar
  26. 26.
    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.PubMedGoogle Scholar
  27. 27.
    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.PubMedCrossRefGoogle Scholar
  28. 28.
    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.PubMedCrossRefGoogle Scholar
  29. 29.
    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.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Therese Sørlie
    • 1
  • Hilde Johnsen
    • 1
  • Phuong Vu
    • 1
  • Guro Elisabeth Lind
    • 1
  • Ragnhild Lothe
    • 1
  • Anne-Lise Børresen-Dale
    • 1
  1. 1.Department of GeneticsThe Norwegian Radium HospitalOsloNorway

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