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Low-Resolution Nucleosome Mapping

Low resolution nucleosome mapping is a technique quite similar to DNA footprinting that employs micrococcal nuclease digestion to localize nucleosomes on a stretch of DNA. Microccocal nuclease is an enzyme that makes double strand cuts in DNA between nucleosomes when incubated with cell nuclei or permeabilized cells. Full digestion with MNase gives mostly mononucleosome size DNA fragments, while partial digests result in single cuts between nucleosomes and fragments of varying size. Generally a range of MNase concentrations are used to produce a collection of cleavage products which can then be run on a gel and probed via southern blot to determine nucleosome position. The quality of MNase analysis is dependent on the integrity of the chromatin substrate and thus use of permeabilized cells is preferable to nuclear isolation. Different information can be obtained by using different concentrations of enzyme. Lower concentrations and partial digests are useful for localizing nucleosomes with respect to a defined point in the DNA, such as a restriction site. Important controls include MNase digestion of protein-free DNA for comparison, since MNase cleaves DNA in a non-random manner. Also, DNA incubated with enzyme buffer but without MNase will demonstrate that cleavage is the result of MNase incubation and not endogenous nucleases. This technique does not give precise position information about nucleosomes but instead indicates the area of the DNA where a nucleosome probably binds.

Editor(s): John Bracht

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