DNA Footprinting - Method

Method

The simplest application of this technique is to assess whether a given protein binds to a region of interest within a DNA molecule. The wet lab methodology is summarized, with appropriate selection of reagents discussed, below.

  1. Polymerase chain reaction (PCR) amplify and label region of interest that contains a potential protein-binding site, ideally amplicon is between 50 to 200 base pairs in length.
  2. Add protein of interest to a portion of the labeled template DNA; a portion should remain separate without protein, for later comparison
  3. 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.
  4. 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.

Read more about this topic:  DNA Footprinting

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