Two-dimensional bacterial genome display: a method for the genomic analysis of mycobacteria

The Division of Infectious and Immunological Diseases, British Columbia’s Children’s Hospital 1 , and Departments of Paediatrics 2 and Pathology & Laboratory Medicine 3 , University of British Columbia, Vancouver, BC, Canada British Columbia Cancer Research Center, 601 West 10th Avenue, Vancouver, BC, V5Z 1L3, Canada 4 Author for correspondence: Edith M. Dullaghan. Tel: +1 604 875 2491. Fax: +1 604 875 2226. e-mail: dullagha{at}interchange.ubc.ca Annually, Mycobacterium tuberculosis is the cause of approximately three million deaths worldwide. It would appear that currently available therapies for this disease are inadequate. The identification of genes involved in mycobacterial virulence will facilitate the design of new prophylactic and therapeutic interventions. A method for high-resolution comparison of bacterial genomes has been developed to facilitate the identification of genes possibly involved in the virulence of clinically relevant mycobacteria. This ‘two-dimensional bacterial genome display’ (2DBGD) method utilizes two-dimensional DNA electrophoresis to separate, on the basis of size and G+C content, genomic fragments generated with different restriction endonucleases. The use of this method to identify genomic differences between species, strains and, most importantly, isogenic mutants of mycobacteria is reported. That 2DBGD can be used to identify differences resulting from either insertional mutagenesis using a gentamicin-resistance gene or from a frameshift mutation is demonstrated. Keywords: Mycobacterium avium complex, tuberculosis, strain differentiation, fingerprint, two-dimensional DNA electrophoresis Abbreviations: 2DBGD, two-dimensional bacterial genome display; 2DDE, two-dimensional DNA electrophoresis; DGGE, denaturing gradient gel electrophoresis; MAC, Mycobacterium avium complex