mtDNA Extraction Research Articles

Isolation and polymorphism in mitochondrial DNA from Schistosoma mansoni

A molecular marker such as mitochondrial DNA (mtDNA), maternally inherited, could provide a tool to better characterize genetic variability in schistosomes (Trematodes, Platyhelminths). MtDNA is a good molecular marker of evolution, especially among closely related species [ 1 ]. Most studies on mtDNA involve vertebrate or arthropod (mainly insect) species, and very few are related to the Nemathelminth [2,3] and Platyhelminth phyla: so far, only the mtDNA of two cestodes, Taenia hydagenata and Echinococcus granulosus [4], and one Trematode, Fasciola hepatica [5], has been studied. The very simple technique we describe here for extraction of mtDNA from Schistosoma mansoni, is based mainly on differential centrifugation and ethanol precipitation. It provides mtDNA pure enough to be vizualized after end-labeling of restriction fragments. Three strains of S. mansoni of human origin: GU (from Guadeloupe, 5 years old), BR (from Brazil, 15 years old), and CI (from Ivory Coast, 2 years old) were maintained in the laboratory in their respective natural vectors, and in mice (Swiss OF 1). Since

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

Publisher Summary 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.