DNA footprinting

DNA footprinting is a method of investigating the sequence specificity of DNA-binding proteins in vitro. This technique can be used to study protein-DNA interactions both outside and within cells.

The regulation of transcription has been studied extensively, and yet there is still much that is not known. Transcription factors and associated proteins that bind promoters, enhancers, or silencers to drive or repress transcription are fundamental to understanding the unique regulation of individual genes within the genome. Techniques like DNA footprinting help elucidate which proteins bind to these associated regions of DNA and unravel the complexities of transcriptional control.

In 1978, David Galas and Albert Schmitz developed the DNA footprinting technique to study the binding specificity of the lac repressor protein. It was originally a modification of the Maxam-Gilbert chemical sequencing technique.

The simplest application of this technique is to assess whether a given protein binds to a region of interest within a DNA molecule.Polymerase chain reaction (PCR) amplify and label region of interest that contains a potential protein-binding site, ideally amplicon is between 50 and 200 base pairs in length. Add protein of interest to a portion of the labeled template DNA; a portion should remain separate without protein, for later comparison. Add a cleavage agent to both portions of DNA template. The cleavage agent is a chemical or enzyme that will cut at random locations in a sequence independent manner. The reaction should occur just long enough to cut each DNA molecule in only one location. A protein that specifically binds a region within the DNA template will protect the DNA it is bound to from the cleavage agent. Run both samples side by side on a polyacrylamide gel electrophoresis. The portion of DNA template without protein will be cut at random locations, and thus when it is run on a gel, will produce a ladder-like distribution. The DNA template with the protein will result in ladder distribution with a break in it, the "footprint", where the DNA has been protected from the cleavage agent. Note: Maxam-Gilbert chemical DNA sequencing can be run alongside the samples on the polyacrylamide gel to allow the prediction of the exact location of ligand binding site.

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