Genotyping of Mycobacterium tuberculosis clinical isolates using IS6110-based restriction fragment length...

A number of phylogenetic studies of Mycobacterium tuberculosis have suggested a highly clonal population structure. Despite the extreme homogeneity of M. tuberculosis strains, the genome is punctuated by a number of polymorphic regions that give rise to sufficient diversity, thus forming the basis for molecular epidemiologic studies of tuberculosis. As such, insertion sequence (IS) 6110, which is unique to members of the M. tuberculosis complex and is present in variable numbers and in discrete genomic locales among strains, has been extensively used in molecular epidemiologic studies. Genotyping, using IS6110-based restriction fragment length polymorphism (RFLP), was standardized by the international community, and this has facilitated inter- and intralaboratory comparison, thereby serving as a model system for subspeciation of M. tuberculosis. When IS6110-based RFLP was used in conjunction with conventional epidemiologic data, its utility was realized. In this chapter, we discuss the basic methodology for conducting IS6110-based RFLP and analyzing the resulting hybridization profiles.