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Construction of Linker-Scanning Mutations by Oligonucleotide Ligation

  • Grace M. Hobson
  • Patricia P. Harlow
  • Pamela A. Benfield
Part of the Methods in Molecular Biology book series (MIMB, volume 31)

Abstract

The purpose of linker-scanning mutagenesis is to create a series of mutant molecules in which individual sections are sequentially replaced with the same “neutral” mutant sequence (see Fig. 1). Typically the technique has been applied to DNA and has most commonly been used to determine the cw-regulatory roles of upstream flanking regions of genes. The technique has several advantages over more random mutational analysis:

Keywords

Alanine Scanning Mutagenesis Synthetic Fragment Mutant Molecule Upstream Flank Region Endonuclease Restriction Site 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    McKnight, S.L. and Kingsbury, R (1982) Transcriptional control signals of a eukaryotic protein-coding gene. Science 217, 316–324PubMedCrossRefGoogle Scholar
  2. 2.
    Cunningham, B.C. and Wells, J.A. (1989) High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science 244, 1081–1085PubMedCrossRefGoogle Scholar
  3. 3.
    Karlsson, O., Edlund, T., Moss, J. B., Rutter, W.J., and Walker, M.D. (1987) A mutational analysis of the insulin gene control region, expression in beta cells is dependent on two related sequences within the enhancer. Proc Natl. Acad. Sci. USA 84, 8819–8823.PubMedCrossRefGoogle Scholar
  4. 4.
    Hobson, G. M., Molloy, G.R., and Benfield, P.A. (1990) Identification of cisacting regulatory elements in the promoter region of the rat brain creatine kinase gene. Mol. Cell. Biol. 10, 6533–6543PubMedGoogle Scholar
  5. 5.
    Bell, L.D., Smith, J.C., Derbyshire, R., Finlay, M., Johnson, I., Gilbert, R., Slocombe, P., Cook, E., Richards, H., Clissold, P, Meredith, D., Powell-Jones, C.H., Dawson, K.M., Carter, B.L, and McCullagh, K.G (1988) Chemical synthesis, cloning and expression in mammalian cells of a gene coding for human tissue-type plasminogen activator. Gene 63, 155–163.PubMedCrossRefGoogle Scholar
  6. 6.
    Dawes, W.J., Miller, J.F., and Ragsdale, C.W. (1988) High efficiency transformation of E coli] by high voltage electroporation. Nucleic Acids Res. 16, 6127–61CrossRefGoogle Scholar
  7. 7.
    Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) In vitro amplification of DNA by the polymerase chain reaction. Molecular Cloning A Laboratory Manual, 2nd ed., Chapter 14. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 14.1–14.35.Google Scholar

Copyright information

© Humana Press Inc, Totowa, NJ 1994

Authors and Affiliations

  • Grace M. Hobson
    • 1
  • Patricia P. Harlow
    • 2
  • Pamela A. Benfield
    • 2
  1. 1.Alfred L DuPont InstituteWilmington
  2. 2.The DuPont Merck Pharmaceutical Company, Experimental StationWilmington

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