Site-Directed Mutagenesis Using a Uracil-Containing Phagemid Template

  • Christian Hagemeier
Part of the Springer Protocols Handbooks book series (SPH)


The ability to introduce specific changes into almost any given DNA sequence has revolutionized the analysis of cloned genes. This technique has enabled researchers to identify regions necessary for the regulation of gene expression. Also, it was and still is instrumental to learn about the importance of functional domains or even single amino acids of proteins. Of the many useful methods available for site-directed mutagenesis, in this chapter I describe a protocol that is based on the “Kunkel Method” (1,2). This method allows the generation of point mutations, deletions, and insertions for a given DNA sequence with high efficiency. This procedure has been used successfully many times and most usefully to map protein interaction sites within the activation domain of the transcription factor E2F (3).


Synthetic Oligonucleotide Isoamyl Alcohol Reaction Vial Helper Phage Mutagenic Oligonucleotide 
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  1. 1.
    Kunkel, T. A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82, 488–492.PubMedCrossRefGoogle Scholar
  2. 2.
    Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154, 367–382.PubMedCrossRefGoogle Scholar
  3. 3.
    Hagemeier, C., Cook, A., and Kouzarides, T. (1993) The retinoblastoma protein binds E2F residues required for activation in vivo and TBP binding in vitro. Nucleic Acids Res. 21, 4998–5004.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Christian Hagemeier
    • 1
  1. 1.Laboratory of Molecular Biology and Pediatric DiseaseHumboldt UniversityCharité BerlinGermany

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