PREPARATION OF HIGH-MOLECULAR-WEIGHT DNA - APPLICATION TO MYCOBACTERIAL CELLS

A method for isolating high-molecular-weight DNA from bacteria is desc ribed. A special feature of the method is the treatment of whole bacte rial cells with an organic solvent (chloroform-methanol (2:11 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 ce lls such as Mycobacterium snegnatis, M. phlei, and Escherichia coli co ntained highly polymerized DNA, as revealed by pulse-field gel electro phoresis. The size and yield of the DNA obtained from E. coli pretreat ed with the organic solvent were quite similar to that of the DNA obta ined from protoplasts. The results strongly suggest that the organic s olvent pretreatment is effective for extracting very large DNA from ba cterial cells and especially from bacteria whose protoplasts cannot be easily formed. (C) 1994 Academic Press, Inc,