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Classic Protocol
Published in association with Cold Spring Harbor Laboratory Press

Nature Methods - 2, 719 - 720 (2005)
doi:10.1038/nmeth0905-719

Micrococcal nuclease–Southern blot assay

Micrococcal nuclease (MNase) is unique among nucleases in its ability to induce double-strand breaks within nucleosome linker regions, but only single-strand nicks within the nucleosome itself. Because of this property, MNase can be used to determine whether a DNA fragment of interest is within a nucleosome1, 2. In addition, MNase can be used to determine the approximate positions of nucleosomes in a region of DNA, if the nucleosomes are consistently positioned. In brief, cell nuclei are isolated and limiting concentrations of MNase are added to the nuclei, resulting in cleavage at nucleosome linker regions. The genomic DNA is purified and the fragments are separated by agarose gel electrophoresis; the resulting ladder of stained bands corresponds in size to multiples of the nucleosome core plus the linker (approx200 base pairs (bp)). To determine whether a DNA fragment of interest is within a nucleosome, the genomic DNA is subjected to Southern blot analysis. If a probe derived from the DNA fragment hybridizes to the ladder of nucleosomal bands, the fragment may indeed be assembled into nucleosomes. To determine nucleosome positioning, the purified genomic DNA must be cleaved with a restriction enzyme before gel electrophoresis and Southern blot analysis.

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Nature Methods
ISSN: 1548-7091
EISSN: 1548-7105
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