Improved preparation of high molecular weight DNA for pulsed-field gel electrophoresis from mycobacteria

Molecular typing is now widely used to aid and supplement conventional epid emiological studies of mycobacterial diseases. Pulsed-field gel electrophor esis (PFGE), in which the entire genome can be represented as a distinct pa ttern of DNA restriction fragments, is a particularly powerful tool in epid emiology for the determination of clonal identity of bacteria providing inf ormation for understanding and controlling the spread of disease. Applicati on of PFGE to the study of mycobacterial diseases has been limited because isolation of high-quality genomic DNA from mycobacterial sources has proved problematic. Here we report a simple, highly effective method for the prep aration of high molecular weight DNA from a range of mycobacterial species. Cultures are continuously stirred and are homogeneous. This enables accura te quantification. The presence of detergent in buffers keeps the cells in suspension throughout preparation enabling efficient lysis. In addition, it is compatible with heat-inactivation of pathogenic mycobacteria and all of the preparation procedures can be carried out with a category III facility . This standardised method of preparation of DNA from mycobacteria means th at PFGE should now be evaluated as a method for typing these organisms and it may be particularly important as a means of typing less well-characteris ed mycobacteria for which other techniques are not available. (C) 2001 Else vier Science B.V. All rights reserved.