DNA Footprinting

DNA footprinting is based on the observation that when a protein binds to DNA, the DNA is protected from chemicals that would otherwise cleave it. In a typical DNA footprinting experiment, a DNA fragment with a suspected protein-binding site is first isolated, then labeled with a radioactive nucleotide or other chemical that will allow it to be detected later on.

Once labeled, the DNA is then mixed in a test tube with a DNA-binding protein and a chemical that cleaves DNA, such as the enzyme DNase I. In a separate test tube, more of the same labeled DNA is mixed with the same cleaving chemical, but without the binding protein. The DNA fragments in each tube are allowed to incubate long enough for the molecule to cleave once, and then are separated by size (fractionated) in a DNA sequencing gel.

The reactions in the two test tubes (one with the binding protein and one without) are then compared. If the DNA actually contains protein-binding sites, these will have been protected from cleaving in the test tube that contains DNA-binding protein, and a "footprint" of those sites where no DNA cleavage occurred will be observed. By comparison with a sequencing reaction run on the same gel, one can determine the exact location where a protein has been bound to the DNA. A related technique, called gel retardation, can also be used to test for protein binding to DNA, but this method is less precise than DNA footprinting.

The protein protects the binding site region from cleavage by DNase or another treatment.