Preparation of high-molecular-weight DNA: application to mycobacterial cells.
A method for isolating high-molecular-weight DNA from bacteria is described. A special feature of the method is the treatment of whole bacterial cells with an organic solvent (chloroform-methanol (2:1, v/v) or ethanol-ether (1:1, v/v)) prior to DNA extraction from the cells. The DNA preparations obtained from organic solvent-pretreated bacterial cells such as Mycobacterium smegmatis, M. phlei, and Escherichia coli contained highly polymerized DNA, as revealed by pulse-field gel electrophoresis. The size and yield of the DNA obtained from E. coli pretreated with the organic solvent were quite similar to that of the DNA obtained from protoplasts. The results strongly suggest that the organic solvent pretreatment is effective for extracting very large DNA from bacterial cells and especially from bacteria whose protoplasts cannot be easily formed.