Isolation of DNA from filamentous fungi and separation into nuclear, mitochondrial, ribosomal, and plasmid components

Abstract

A general procedure for purifying and efficiently separating four types of DNA from filamentous fungi has been developed. The protocol involves (i) disruption of mycelial cells by blending in liquid nitrogen followed by suspension of cell contents in buffer containing high concentrations of protease and EDTA; (ii) deproteinization with phenol; (iii) cesium chloride/bisbenzimide density gradient centrifugation to separate nuclear DNA, mitochondrial DNA, and ribosomal DNA; and (iv) agarose gel electrophoresis to identify and purify plasmid DNA, if present. All DNAs are suitable for digestion with restriction endonucleases, ligation, and cloning in Escherichia coli, and DNAs from step three are recovered in high-molecular-weight form. The procedure has been used successfully with several dozen isolates of the plant pathogenic fungus Cochliobolus heterostrophus (including both laboratory strains and isolates collected directly from the field), and has been found to be equally suitable for C. carbonum, Neurospora crassa, N. tetrasperma, and Nectria haematococca.