text: A variety of cleavage agents can be chosen.   a desirable agent is one that is sequence neutral, easy to use, and is easy to control.  Unfortunately no available agents meet all of these standards, so an appropriate agent can be chosen, depending on your DNA sequence and ligand of interest.  The following cleavage agents are described in detail:
  DNase I is a large protein that functions as a double-strand endonuclease.  It binds the minor groove of DNA and cleaves the phosphodiester backbone.  It is a good cleavage agent for footprinting because its size makes it easily physically hindered.  Thus is more likely to have its action blocked by a bound protein on a DNA sequence.  In addition, the DNase I enzyme is easily controlled by adding EDTA to stop the reaction.  There are however some limitations in using DNase I.  The enzyme does not cut DNA randomly; its activity is affected by local DNA structure and sequence and therefore results in an uneven ladder.  This can limit the precision of predicting a protein’s binding site on the DNA molecule.
  Hydroxyl radicals are created from the Fenton reaction, which involves reducing Fe with HO to form free hydroxyl molecules.  These hydroxyl molecules react with the DNA backbone, resulting in a break.  Due to their small size, the resulting DNA footprint has high resolution.  Unlike DNase I they have no sequence dependence and result in a much more evenly distributed ladder.  The negative aspect of using hydroxyl radicals is that they are more time consuming to use, due to a slower reaction and digestion time.
  Ultraviolet irradiation can be used to excite nucleic acids and create photoreactions, which results in damaged bases in the DNA strand.  Photoreactions can include: single strand breaks, interactions between or within DNA strands, reactions with solvents, or crosslinks with proteins. The workflow for this method has an additional step, once both your protected and unprotected DNA have been treated, there is subsequent primer extension of the cleaved products.  The extension will terminate upon reaching a damaged base, and thus when the PCR products are run side-by-side on a gel; the protected sample will show an additional band where the DNA was crosslinked with a bound protein. Advantages of using UV are that it reacts very quickly and can therefore capture interactions that are only momentary.  Additionally it can be applied to in vivo experiments, because UV can penetrate cell membranes.  A disadvantage is that the gel can be difficult to interpret, as the bound protein does not protect the DNA, it merely alters the photoreactions in the vicinity.