In 1999, the virulence genes associated with Mycobacterium tuberculosis were identified through transposon mutagenesis-mediated gene knockout. A plasmid named pCG113 containing kanamycin resistance genes and the IS1096 insertion sequence was engineered to contain variable 80-base pair tags. The plasmids were then transformed into M. tuberculosis cells by electroporation. Colonies were plated on kanamycin to select for resistant cells. Colonies that underwent random transposition events were identified by BamHI digestion and Southern blotting using an internal IS1096 DNA probe. Colonies were screened for attenuated multiplication to identify colonies with mutations in candidate virulence genes. Mutations leading to an attenuated phenotype were mapped by amplification of adjacent regions to the IS1096 sequences and compared with the published M. tuberculosis genome. In this instance transposon mutagenesis identified 13 pathogenic loci in the M. tuberculosis genome which were not previously associated with disease. This is essential information in understanding the infectious cycle of the bacterium.