Identification of new markers by genome wide comparison

AMADIO A; ROMANO MI; CATALDI A; CAIMI K

Florianopolis, Brasil

Workshop; Second General Meeting INCO-DEV Concerted Action. “Improved Diagnosis, Drug Resistance Detection and Control of Tuberculosis in Latin America”; 2003

Introduction: Insertion/deletion (in/del) events seem to play an essential role in M. tuberculosis complex speciation and intraspecies diversity. In/del has been proposed as a method for typing of M. tuberculosis. On the other hand genome comparisons are emerging as a powerful tool to identify gene families and to infer evolutionary relationships. Methods: a genome-wide in silico comparison between M. tuberculosis H37Rv and M. bovis AF2122/97has allowed us to identify several regions of difference between this two M. tuberculosis complex species. SNP were not analyzed in this work. Several of these In/del are already described while other are novel. The comparison was made using Blastn and MSPCrunch software and visualized using Artemis Comparison Tool (ACT). The regions of difference, if not reported in the literature, were then confirmed by Blast searches against M. bovis genome and subsequently confirmed by PCR using pairs of primers external and internal to the deletions. Results: We observed 23 regions of difference. Some of them involving several genes while other comprising the deletion of part of a gene. 18 of them were already described. While five are novel In/del fragments. We characterized some of them RV0647-8 (intergenic region), Rv0867 and Rv3479. These marker genes showed to be variable between different isolates of the M. tuberculosis complex. Discussion: the novel markers identified in this work may be useful for intra or inter species differentiation. Other important aspect to be studied would be to determine the function of these genes and their role in phenotypic properties of the M. tuberculosis complex species.