DNase footprinting assay

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DNaseI footprint of a protein binding to a radiolabelled DNA fragment. Lanes "GA" and "TC" are Maxam-Gilbert chemical sequencing lanes, see DNA_sequencing. The lane labelled "control" is for quality control purposes and contains the DNA fragment but not treated with DNaseI.

A DNase footprinting assay [1]is a technique from molecular biology that detects DNA-protein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage. The method uses an enzyme, deoxyribonuclease (DNase, for short) to cut the radioactively end-labelled DNA, followed by gel electrophoresis to detect the resulting cleavage pattern.

For example, the DNA fragment of interest may be PCR amplified using a ³²P 5' labelled primer, with the end 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 electropheretic gel. The fragments which are smaller with respect to the ³²P-labelled end 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.