Chapter 16 The Use of Fluorescent DNA-Binding Agent for Detecting and Separating Yeast Mitochondrial DNA

Abstract

This chapter focuses on use of fluorescent DNA-binding agent for detecting and separating yeast mitochondrial DNA (mtDNA). There are three main routes to the isolation of pure mtDNA. The first approach involves chromatographic separation of the nuclear and mitochondrial components from extracts of broken cells or protoplasts. Examples are the hydroxyapatite column procedure and the polylysine–kieselguhr system. Both these techniques are potentially capable of delivering large yields, but they necessarily involve considerable mechanical degradation and their scale makes them unsuitable for the routine detection of small amounts. Similar criticisms may be leveled at the second approach to this problem that involves the extraction of mtDNA from isolated mitochondria. Isolation of the organelles is a difficult enough task and they are heavily contaminated with nuclear DNA whose elimination is a serious problem. The cesium chloride gradient procedure offers one outstanding advantage in dealing with what appears to be a remarkably labile species of DNA—namely, the potential for minimizing both mechanical and enzymatic degradation. In the chapter, the use of 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) in cesium chloride gradients, fluorescent staining of cells with DAPI, and sensitivity of the staining procedure are discussed. © 1975, Academic Press, Inc.