“DNase footprinting assay”的意思、由来-开放百科全书

A DNase footprinting assay^[1] is a DNA footprinting 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 PCR amplified using a ³²P 5' labeled primer, 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 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.

This technique was developed by David Galas and Albert Schmitz at Geneva in 1977^[2]

See also

References

1. ^{{cite journal |doi=10.1016/0076-6879(86)30011-9 |vauthors=Brenowitz M, Senear DF, Shea MA, Ackers GK |title=Quantitative DNase footprint titration: a method for studying protein-DNA interactions |journal=Methods in Enzymology |volume=130 |issue= |pages=132–81 |year=1986 |pmid=3773731 }}
2. ^{{cite journal |vauthors=Galas DJ, Schmitz A |title=DNAse footprinting: a simple method for the detection of protein-DNA binding specificity |journal=Nucleic Acids Research |volume=5 |issue=9 |pages=3157–70 |date=Sep 1978 |pmid=212715 |pmc=342238 |doi= 10.1093/nar/5.9.3157|url=http://nar.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=212715}}