Abstract
This chapter describes the techniques necessary to identify p53 genomic mutations in either frozen or paraffin-embedded tumor samples. DNA is extracted from the tumor samples and then used as a template to amplify the p53 coding sequences by polymerase chain reaction (PCR). The PCR products are characterized by agarose gel electrophoresis. Subsequently, mutations can be detected by direct DNA sequencing of the PCR products, followed by acrylamide gel electrophoresis and autoradiography. Alternatively, PCR products containing mutations can be identified by their aberrant mobility on denaturing acrylamide gels, using single-stranded conformation polymorphism (SSCP).
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
Kerr, J. F., Wylie, A. H., and Currie, A. R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257.
Vousden, K. H. (2000) p53: death star. Cell 103, 691–694.
Moll, U. M. and Zaika, A. (2001) Nuclear and mitochondrial apoptotic pathways of p53. FEBS Lett. 493, 65–69.
Hollstein, M., Hergenhahn, M., Yang, Q., Bartsch, H., Wang, Z. Q., and Hainaut, P. (1999) New approaches to understanding p53 gene tumor mutation spectra. Mutat. Res. 431, 199–209.
Hainaut, P. and Hollstein, M. (2000) p53 and human cancer: the first thousand mutations. Adv. Cancer Res. 77, 81–137.
Orita, M., Suzuki, Y., Sekiya T., and Hayashi K. (1989) Rapid and sensitive method of point mutation and DNA polymorphisms using polymerase chain reaction. Genomics 5, 874–879.
Sambrook, J. and Russell, D. W. (eds.) (2001) Molecular Cloning. A Laboroatory Manual. CSH Laboratory Press, Cold Spring Harbor, NY.
Ausubel, F. M. (1999) Current Protocols in Molecular Biology. Wiley, New York.
Lu, X., Park, S. H., Thompson, T. C., and Lane, D. P. (1992) ras-induced hyperplasia occurs with mutation of p53, but activated ras and myc together can induce carcinoma without p53 mutation. Cell 70, 153–161.
Frank, T. S, Svoboda-Newman, S. M., and Hsi, E. D. (1996) Comparison of methods for extracting DNA from formalin-fixed paraffin sections for nonisotopic PCR. Diagn. Mol. Pathol. 5, 220–224.
Hollstein, M., Sidransky, D., Vogelstein, B., and Harris, C. C. (1991) p53 mutations in human cancers. Science 253, 49–53.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Erster, S., Slade, N., Moll, U.M. (2003). Mutational Analysis of p53 in Human Tumors. In: Deb, S., Deb, S.P. (eds) p53 Protocols. Methods in Molecular Biology, vol 234. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-408-5:219
Download citation
DOI: https://doi.org/10.1385/1-59259-408-5:219
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-1-58829-106-6
Online ISBN: 978-1-59259-408-5
eBook Packages: Springer Protocols