Nurturing Life Sciences
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#Unit 13. Methods in Biology
DNase footprinting gives a good idea of the location of the binding site for the protein, but DNase is a macromolecule and is therefore a rather blunt instrument for probing the fine details of the binding site.
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DNA-binding proteins frequently perturb the DNA within the binding region, distorting the double helix. These perturbations are interesting, but are not generally detected by DNase footprinting because the protein keeps the DNase away.
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More detailed footprinting requires a smaller molecule that canfit into the nooks and crannies of the DNA–protein complex and reveal more of the subtleties of the interaction. A favorite tool for this job is the methylating agent dimethyl sulfate (DMS).
More detailed footprinting requires a smaller molecule that can
fit into the nooks and crannies of the DNA–protein complex and reveal more of the subtleties of the interaction. A favorite tool for this job is the methylating agent dimethyl sulfate (DMS).
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DMS footprinting follows a similar principle, except that we use the DNA methylating agent DMS, instead of DNase, to attack the DNA–protein complex.
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In DMS footprinting, Unmethylated (or hypermethylated) sites show up on electrophoresis and demonstrate where the protein is bound to the DNA.
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Methylation interference assay Dimethyl sulphate (DMS) methylates purines on DNA strand. Methylate DNA prior to adding protein.Protein binding is prevented at methylated sites