DNA Footprinting

DNA footprinting is a technique for identifying exactly where a protein binds to DNA. Knowing where a protein binds to DNA often aids in understanding how gene expression is regulated. Consequently, DNA footprinting is often part of a larger study to determine how a particular gene is controlled.

How It Works

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.

Uses in Research

DNA footprinting is often used to locate the binding site for proteins that regulate transcription. For example, a researcher may suspect that a particular protein binds to a particular DNA fragment and inhibits transcription. After conducting a DNA footprinting experiment, the researcher will know the location of the exact sequence of DNA bound by that protein. If that sequence matches the sequence of a promoter the DNA footprinting experiment can help explain how that DNA-binding protein carries out its function.

Modified DNA footprinting experiments can also be performed to detect where proteins bind to DNA in a living cell. In these experiments, cells are grown under conditions where the protein of interest would be expected to bind to DNA. The cells are then treated with a chemical that causes proteins bound to DNA to become permanently attached to the DNA. The resulting DNA-protein complexes are then purified from the cell, and the DNA sequences are identified.

Since DNA footprinting is used to identify the specific sequences in DNA where a protein binds, the technique is likely to be of continuing usefulness in genetic research. For example, DNA footprinting is likely to be heavily used in characterizing the function of proteins identified in the Human Genome Project and other genome projects, making it an important component of a molecular geneticist's toolbox.

Patrick G. Guilfoile

Bibliography

Guilfoile, Patrick. A Photographic Atlas for the Molecular Biology Laboratory. Englewood, CO: Morton Publishing, 2000.

Internet Resource

DNA Footprinting Reveals the Sites Where Proteins Bind on a DNA Molecule. National Center for Biotechnology Information. <http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books>.