7.25H: DNA Protection Analysis
DNA protection or “footprinting” analysis is a powerful technique for identifying the nucleotides involved in a protein-DNA interaction.
LEARNING OBJECTIVES
Illustrate DNA protection analysis
KEY TAKEAWAYS
DNA protection or footprinting is a technique from molecular biology/biochemistry that detects DNA-protein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage. This makes it possible to locate a protein binding site on a particular DNA molecule. The method uses an enzyme, deoxyribonuclease (DNase, for short) to cut the radioactively end-labeled DNA, followed by gel electrophoresis to detect the resulting cleavage pattern. For example, the DNA fragment of interest may be amplified by polymerase chain reaction, with the result being many DNA molecules with a radioactive label on one end of one strand of each double stranded molecule. Cleavage by DNase will produce fragments, the smaller of which will move further on the electrophoretic gel.
The fragments which are smaller will appear further on the gel than the longer fragments. The gel is then used to expose a special photographic film. The cleavage pattern of the DNA in the absence of a DNA binding protein, typically referred to as free DNA, is compared to the cleavage pattern of DNA in the presence of a DNA binding protein. If the protein binds DNA, the binding site is protected from enzymatic cleavage. This protection will result in a clear area on the gel which is referred to as the “footprint”. By varying the concentration of the DNA-binding protein, the binding affinity of the protein can be estimated according to the minimum concentration of protein at which a footprint is observed. This technique was developed by David Galas and Albert Schmitz at Geneva in 1977.
Key Points
- DNA protection analysis is a technique in which a DNA molecule is ‘incubated’ with a protein that binds to a specific site along the double helix.
- The DNA-binding protein complex is then subjected to restriction endonuclease digestion, which reduces the entire DNA to mono- and oligonucleotide fragments, except for the portion of the DNA molecule that was ‘protected’ from digestion by the binding protein.
- Removal of the protein by simple chemical means—e.g., by gel electrophoresis —allows the study of DNA and binding protein interaction.
Key Terms
- electrophoresis : a method for the separation and analysis of large molecules (such as proteins) by migrating a colloidal solution of them through a gel; gel electrophoresis
- polymerase chain reaction : A technique in molecular biology for creating multiple copies of DNA from a sample; used in genetic fingerprinting etc.