As pointed out earlier, the binding site on DNA for lac repressor was characterized by filter binding assay (Fig. 35.11). However, the technique of footprinting has been extensively used for identification of DNA sites used for binding specific proteins leading to regulation of gene expression. Following steps are followed using two samples of DNA to be examined (one of them used as control and the other complexed with protein, whose DNA binding sites need to be determined) : (i) DNA is end labelled; (ii) labelled DNA samples are subjected to partial cleavage (so that all cleavage sites are not attacked) with DNAase I; (iii) population of nicked DNA molecules in both samples recovered by removing protein from the complex; (iv) DNA segments are denatured; (v) DNA segments in both samples are run in parallel for electrophoretic separation. A comparison of electrophoretic patterns in two samples will show a number of bands missing in the gel, where DNA-protein complex was used as a sample. The missing bands represent the position of DNA sites protected by protein and will allow the determination of DNA region used by protein for binding (Fig. 35.20). If above samples are treated for sequencing, the sequence of binding site can be directly 'read off from the gel (see sequencing methods in Genetic Engineering and Biotechnology 3.  Isolation, Sequencing and Synthesis of Genes).