Abstract
Transcriptional control is mediated through interactions involving specific nuclear proteins and target DNA sequences found in the promoters and other regulatory regions of eukaryotic genes. Consequently, the ability to define the location and characterize the nature of these interactions is central to our understanding of gene regulation. Although a number of different techniques are available that allow the detection of DNA-protein complexes, one method that has emerged to be the most useful is DNase I footprinting. This technique allows the investigator not only to detect DNA-protein complexes, but also to define precisely the boundaries of these interactions within the DNA molecule.
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References
Galas, D. J. and Schmitz, A. (1978) DNase footprinting: a simple method for the detection of protein-DNA-binding specificity. Nucleic Acids Res. 5, 3157–3170.
Murray, E. E., Lewis, K., Sobol, M., and Binghui, L. (1992) Optimizing and troubleshooting DNase 1 footprinting reactions. Promega Notes 36, 1–11.
Higuchi, R., Krummel, B., and Saiki, R. (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16, 7351–7367.
Hooft van Huijsduijnen, R. A. M. (1992) PCR-generated probes for the study of DNA-protein interactions. BioTechniques 12, 830–832.
Iannello, R. C, Kola, I., and Hans-Henrik, M. D. (1993) Temporal and tissuespecific interactions involving novel transcription factors and the proximal promoter of the mouse Pdha-2 gene. J. Biol. Chem. 268, 22,581–22,590.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Sawadogo, M. and Van Dyke, M. W. (1991) A rapid method for the purification of deprotected oligodeoxynucleotides. Nucleic Acids Res. 19, 674.
Gorski, K., Carneiro, M., and Schibler, U. (1986) Tissue-specific in vitro transcription from the mouse albumin promoter. Cell 47, 767–776.
Tamura, T.-A., Ohya, Y., Miura, M., Aoyama, A., Inoue, T., and Mikoshiba, K. (1989) Preparation of large amounts of transcription-competent nuclear extracts from mouse brain. Technique 1, 33–36.
Mar, J. H. and Ordahl, C. P. (1990) MCAT binding factor, a novel trans-acting factor governing muscle-specific transcription. Mol. Cell. Biol. 10, 4271–4283.
Iannello, R. C, Mar, J. H., and Ordahl, C. P. (1991) Characterization of a promoter element required for transcription in myocardial cells. J. Biol. Chem. 266, 3309–3316.
Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489.
Maxim, A. M. and Gilbert, W. (1980) Sequencing end-labelled DNA with basespecific cleavages. Methods Enzymol. 65, 499–560.
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© 1995 Humana Press Inc.
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Iannello, R.C. (1995). DNase i footprinting using PCR-generated end-labeled DNA probes. In: Tymms, M.J. (eds) In Vitro Transcription and Translation Protocols. Methods in Molecular Biology, vol 37. Humana Press. https://doi.org/10.1385/0-89603-288-4:379
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DOI: https://doi.org/10.1385/0-89603-288-4:379
Publisher Name: Humana Press
Print ISBN: 978-0-89603-288-0
Online ISBN: 978-1-59259-524-2
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