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A collection of useful molecular techniques to illuminate and explore the interaction of drugs and ligands with DNA. These easily reproducible methods involve sequence recognition properties, as well as the physical approaches for measuring both the strength of interaction and the mode of drug binding to DNA. The interactions are also examined from a cellular perspective and for their usefulness in the design of new therapeutic agents. The powerful techniques detailed here will be particularly useful in elucidating the action of existing therapeutic agents, as well as in the design of new anti-cancer drugs with improved action and reduced toxicity.
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1 DNase I Footprinting Keith R. Fox 1. Introduction Footprmtmg provides a simple, quick, and reasonably mexpensive method for assessingthe sequence specific mteraction of ligands with DNA. Although the techmque was developed in 1978 for studying the mteraction of DNAbinding proteins with then target sites (I), it has proved invaluable for determining the sequence specificity of many small hgands 1.1. Footprinting , Footprmting is essentially a protection assay, m which cleavage of DNA is inhibited at discrete locations by the sequence specific binding of a hgand or protein. In this technique, a DNA fragment of known sequence and length (typically a restriction fragment of 100-200 bp), which has been selectively radiolabeled at one end of one strand, IS lightly dtgested by a suitable endonucleolytic probe m the presence and absence of the drug under investigation The cleavage agent is prevented from cutting around the drug-binding sites so that, when the products of reaction are separated on a denaturing polyacrylamide gel and exposed to autoradiography, the position of the ligand can be seen as a gap m the otherwise continuous ladder of bands (see Fig. 1). In this figure, cleavage at position “a” will produce, afte