Theoretical analysis of the footprinting experiment.

In the footprinting experiment, an end-radiolabeled DNA restriction fragment is subjected to digest by an endonuclease in the presence and absence of a ligand which alters the endonuclease cleavage rate at sites of ligand-DNA contact. The location of these sites, and the strength of the ligand binding, are then deduced from the measured concentrations of the different oligonucleotides produced by the digest. We analyze the experiment in terms of coupled kinetic equations which take into account the cutting rates of endonuclease for sites with ligand present and absent, and the rates of binding and dissociation of the ligand to a site. As long as the ligand concentration remains essentially constant (which occurs, for example, if digest is terminated early enough to assure that all fragments result from single cuts by the endonuclease), the oligonucleotide concentrations reflect only the ligand binding equilibrium constant (ratio of rate constants) and the cutting rates in the presence and absence of ligand. We also show how the measured oligonucleotide concentrations (from, e.g. an autoradiogram) can be used to deduce the ligand equilibrium binding constants for the various sites on the polymer.