Practice question

DNase I footprinting identifies precise binding sites of DNA-binding proteins. End-labeled DNA fragment is partially digested with DNase I, a non-specific endonuclease introducing single-strand nicks at random phosphodiester bonds, generating ladder when resolved on denaturing gel. Pre-incubation with DNA-binding protein protects phosphodiester backbone within contact region from cleavage, preventing production of corresponding fragments. Hence protected region appears as gap, or missing bands, footprint against background ladder. Position of gap defines recognition site, while flanking cleavage pattern provides sequence context for transcription factor binding analysis.