Abstract
The technique of site-directed mutagenesis has been used to characterize gene and protein structure–function relationships, protein–protein interactions, binding domains of proteins, or active sites of enzymes for the last three decades. In this technique, a nucleotide sequence of interest is experimentally altered using synthetic oligonucleotides. The most commonly used approach is to use an oligonucleotide that is complementary to part of a single-stranded DNA template, but containing an internal mismatch to direct the mutation. In addition to single point mutations, this approach may also be used to construct multiple mutations, insertions, or deletions. As a result of its broad applicability in disease gene characterization studies, numerous commercial kits are now available, making this technique quick, straightforward, and reliable. In this chapter, we detail the steps involved in site-directed mutagenesis and highlight the essentials of this versatile technique based upon our experience.
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Acknowledgements
The authors would like to acknowledge Pinar Tuzmen for her critical review and her continued support. Additionally, the authors acknowledge Agilent Technologies, Stratagene Products Division, Life Technologies Corporation, Invitrogen Products Division, and Promega Corporation for their permission to reference their Site Directed Mutagenesis kits, and for the use of protocols, figures, and related information included in the manuals of the kits as the source of the materials and methods.
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Carrigan, P.E., Ballar, P., Tuzmen, S. (2011). Site-Directed Mutagenesis. In: DiStefano, J. (eds) Disease Gene Identification. Methods in Molecular Biology, vol 700. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61737-954-3_8
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DOI: https://doi.org/10.1007/978-1-61737-954-3_8
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-61737-954-3
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