Methods in molecular biology · 2009 · 63 citations · 7 references
Life SciencesBiologyMolecular BiologyEnzymologyGene ExpressionProtein BiochemistryNucleic Acid-protein InteractionsProtein-nucleic Acid InteractionsTranscription FactorsDigestionDna Binding SitesDna-protein RecognitionEnzymatic DigestionInteraction Site MappingProtein FootprintingSequence-specific Dna BindingTranscription Factor Binding Sites
The association of proteins with the DNA double helix can interfere with the accessibility of the latter to nucleases. This is particularly true when using bulky nucleases such as DNAse I. The DNAse I footprinting method was developed to make use of this phenomenon in the study of DNA-protein interactions; it consists in comparing the pattern of fragments produced by the partial digestion of DNA in the absence of a protein to that produced by partial digestion of DNA in the presence of a protein. Normally, when the two sets of fragments are separated side by side on a gel, the fragments produced in the presence of the protein will feature blank regions (indicating protection) and/or enhanced cleavage sites (indicating increased availability). This technique can furthermore reveal if multiple sites for a DNA-binding protein are present on a same fragment, and allow the comparison of their respective affinities.
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