Abstract
Site-directed mutagenesis is a powerful tool used in modern molecular biology for protein and genetic engineering. A number of simple and elegant protocols are available to introduce mutations into a target DNA sequence. However, there are only a few methods described for deletion mutagenesis (1–4). In this chapter, we demonstrate a simple method for creating internal deletions into any desired position in a target DNA sequence. The procedure involves replacement of the DNA region to be deleted with a restriction site for an endonuclease. This also facilitates the insertion of other DNA sequences into the site or if desired restoration of the deleted fragment. Introduction of the new restriction site also provides a useful diagnostic tool for screening for positive clones following the transformation procedure.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Legerski, R. J., Hodnett, J. L., and Gray, H. B., Jr. (1978) Extracellular nucleases of pseudomonas BAL 31. III. Use of the double-strand deoxyribonuclease activity as the basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzymes. Nucleic Acid Res. 5, 1445–1464.
Sakonju S., Bogenhagen, D. F., and Brown, D. D. (1980) A control region in the center of the 5S RNA gene directs specific initiation of transcription: I. The 5′ border of the region. Cell 19, 13–25.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
King, P. and Goodbourn, S. (1992) A method for sequence-specific deletion mutagenesis. Nucleic Acid Res. 20, 1039–1044.
Ohtsuki, M., Tomic-Canic, M., Freedberg, I. M., and Blumenberg, M. (1992) Nuclear proteins involved in transcription of the human K5 keratin gene. J. Invest. Dermatol. 99, 206–215.
Bernerd, F., Magnaldo, T., Freedberg, I. M., and Blumenberg, M. (1993) Expression of the carcinoma-associated keratin K6 and the role of AP-1 proto-oncoproteins. Gene Expression 3, 187–199.
Erlich, M. (ed.) (1989) PCR Technology: Principles and Applications for DNA Amplification, Stockton Press, New York.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Humana Press Inc.
About this protocol
Cite this protocol
Tomic-Canic, M., Bernerd, F., Blumenberg, M. (1996). A Simple Method to Introduce Internal Deletions or Mutations into Any Position of a Target DNA Sequence. In: Trower, M.K. (eds) In Vitro Mutagenesis Protocols. Methods In Molecular Medicine™, vol 57. Humana Press. https://doi.org/10.1385/0-89603-332-5:249
Download citation
DOI: https://doi.org/10.1385/0-89603-332-5:249
Publisher Name: Humana Press
Print ISBN: 978-0-89603-332-0
Online ISBN: 978-1-59259-544-0
eBook Packages: Springer Protocols