Detection of Sequence-Specific Protein-DNA Interactions by the DNA-Footprinting Technique

  • Mark A. Plumb
  • Graham H. Goodwin
Part of the Methods in Molecular Biology book series (MIMB, volume 4)


The initiation of transcription of eukaryotic genes by RNA polymerases is controlled by complex interactions between nonhistone proteins and specific regulatory DNA sequences (promoters and enhancers) (1). In order to characterize and purify such transacting protein factors, a sensitive and accurate assay for sequence-specific DNA-binding proteins is the DNA-footprinting technique, which can be used to analyze the interaction of a complex mixture of proteins with a gene regulatory sequence(s) that is known to be important for the expression of that gene.


Storage Buffer Nonhistone Protein Calf Intestinal Phosphatase Bacterial Alkaline Phosphatase Nonspecific Competitor 
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  1. 1.
    Dynan, W.S. and Tjian, R. (1985) Control of eukaryotic messenger RNA synthesis by sequence-specific DNA-binding proteins. Nature 316, 774–778.PubMedCrossRefGoogle Scholar
  2. 2.
    Maxam, A.M. and Gilbert, W. (1977) A new method for sequencing DNA. Proc. Natl. Acad. Set. USA 74, 560–564.CrossRefGoogle Scholar
  3. 3.
    Van Dyke, M.W. and Dervan, P.B. (1983) Methidiumpropyl-EDTA-Fe(11) and DNAse1 footprinting report different small molecule binding site sizes on DNA. Nucl. Acids Res. 11, 5555.PubMedCrossRefGoogle Scholar
  4. 4.
    Wu, C. (1985) An exonuclease protection assay reveals heat-shock element and TATA box DNA-binding proteins in crude nuclear extracts. Nature 317, 84–87.PubMedCrossRefGoogle Scholar
  5. 5.
    Von der Ahe, D., Renoir, J.M., Buchon, T., Baulieu, E.-E., and Beato, M. (1986) Receptors for glucocorticosteroid and progesterone recognise distinct features of a DNA regulatory element. Proc. Natl. Acad. Sci. USA 83, 2817–2821.PubMedCrossRefGoogle Scholar
  6. 6.
    Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) inMolecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.Google Scholar
  7. 7.
    Berkner, K.L. and Folk, W.R. (1977) Polynucleotide kinase exchange reaction. J. Biol. Chem. 252, 3176–3184.PubMedGoogle Scholar
  8. 8.
    Berkner, K.L. and Folk, W.R. (1979) Quantitation of the various termini generated by type II restriction endonucleases using the polynucleotide kinase exchange reaction. J. Biol. Chem. 254, 2561–2564.PubMedGoogle Scholar
  9. 9.
    Tu, C-P.D. and Cohen, S.N. (1980) 3′-End labelling of DNA with [α-32P] cordycepin-5′-triphosphate. Gene 10, 177–183.PubMedCrossRefGoogle Scholar
  10. 10.
    Roychoudhury, R., Tu, C-P.D., and Wu, R. (1979) Influence of nucleotide sequences adjacent to duplex DNA termini on 3′ terminal labelling by terminal transferase. Nucleic Acids Res. 6, 1323–1333.PubMedCrossRefGoogle Scholar
  11. 11.
    Vierira, J. and Messing, J. (1982) The pUC plasmids, and M13 mp 7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 10, 259–268.CrossRefGoogle Scholar
  12. 12.
    Plumb, M.A., Nicolas, R.H., Wright, CA., and Goodwin, G.H. (1985) Multiple sequence-specific DNA binding activities are eluted from chicken nuclei at low ionic strength. Nucleic Acids Res. 13, 4047–4065.PubMedCrossRefGoogle Scholar
  13. 13.
    Emerson, B.M., Lewis, CD., and Felsenfeld, G. (1985) Interaction of specific nuclear factors with the nuclease-hypersensitive region of the chicken adult β-globin gene: Nature of the binding domain. Cell 41, 21–30.PubMedCrossRefGoogle Scholar
  14. 14.
    Emerson, B.M. and Felsenfeld, G. (1984) Specific factor conferring nuclease hypersensitivity at the 5′ end of the chicken adult β-globin gene. Proc. Natl. Acad. Sci. USA 81, 95–99.PubMedCrossRefGoogle Scholar
  15. 15.
    Dolan, M., Dodgson, J.B., and Engel, J.D. (1983) Analysis of the adult chicken β-globin gene. J. Biol. Chem. 258, 3983–3990.PubMedGoogle Scholar
  16. 16.
    Clewell, D. and Helinski, D.R. (1970) Properties of supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of relaxation event. Biochemistry 9, 4428–4440.PubMedCrossRefGoogle Scholar

Copyright information

© The Humana Press Inc. 1988

Authors and Affiliations

  • Mark A. Plumb
    • 1
  • Graham H. Goodwin
    • 2
  1. 1.Beatson Institute for Cancer ResearchGlasgowUK
  2. 2.Chester Beatty LaboratoriesInstitute of Cancer ResearchLondonUK

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