DNase I Footprinting

DNase I footprinting has found a wide following for both identifying and characterizing DNA–protein interactions, particularly because of its simplicity. The concept is that a partial digestion by DNase I of a uniquely 32 P-end-labeled fragment will generate a ladder of fragments, whose mobilities on a denaturing acrylamide gel and whose positions in a subsequent autoradiograph will represent the distance from the end label to the points of cleavage. Bound protein prevents binding of DNase I in and around its binding site and thus generates a “footprint” in the cleavage ladder. The distance from the end label to the edges of the footprint represents the position of the protein-binding site on the DNA fragment. The position of the binding site can be determined by electrophoresing a DNA sequencing ladder alongside the footprint. DNase I cannot bind directly adjacent to a DNA-bound protein because of steric hindrance. Hence, the footprint gives a broad indication of the binding site, generally 8–10 base pairs (bp) larger than the site itself.