Protocol

Micrococcal Nuclease-Southern Blot Assay: I. MNase and Restriction Digestions

This protocol was adapted from "In Vivo Analysis of an Endogenous Control Region," Chapter 10, in Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, 1st edition, by Michael Carey and Stephen T. Smale. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000.

INTRODUCTION

This is the first phase of a two-part assay for exploring whether an endogenous control region of interest is assembled into nucleosomes or is devoid of nucleosomes. This assay can also be used to determine if nucleosomes are similarly positioned at the locus in every cell within a population. Micrococcal nuclease (MNase) is unique among nucleases in its relative ability to induce double-strand breaks within nucleosomal linker regions, but only single-stranded nicks within the nucleosome itself. Because of this property, micrococcal nuclease can be used to determine whether a DNA fragment of interest is nucleosomal. In this phase of the assay, the cells are lysed and nuclei are isolated. Limiting concentrations of micrococcal nuclease are added to the nuclei, resulting in cleavage at nucleosome linker regions (preferably cleavage at two sites per DNA molecule). The cleavage reactions are stopped, and then the genomic DNA is purified and digested with restriction enzymes.