Background.

The biological differences among members of the Mycobacterium avium complex (MAC) are undoubtedly encoded in their genomic sequence. Newly-developed DNA microarray technology along with sequence data from the M. avium subsp. paratuberculosis (MAP) and M. avium avium (MAA) projects together provide the opportunity to assemble a MAC whole genome DNA microarray.

Design.

Exploiting the inherent gene homology between M. tuberculosis (MTB) and MAC, we have acquired a set of synthetic 70bp oligonucleotides that represent each of the 3924 open reading frames (ORFs) of MTB. To this set, we have added custom-designed oligonucleotides for 647 ORFs that are either unique to MAA (no homology to MTB) or present in MAP but absent from MAA. These combined oligonucleotide sets are printed together to create the first generation MAC DNA microarray.

Validation.

In preliminary experiments, we have validated the use of oligonucleotide probes for the GC-rich MAC. Probes have been designed for homologues of MTB genes known to be induced by isoniazid. Gene expression profiling using this array yields results consistent with those seen by semi-quantitative RT-PCR. In ongoing experiments, MAA DNA is applied to the combined array in parallel with MTB DNA: for each gene representative, the intensity of the two signals is plotted against the degree of DNA homology of the 70 base-pair probe between the 2 species. A homology cut-off point below which hybridization intensity is inadequate is thus determined. For these genes, oligonucleotides specific to MAA are designed and used in the construction of the second generation array.

Conclusion.

The assembly and validation of a whole-genome oligonucleotide microarray is being carried out. This tool will enable comprehensive comparative and functional genomic studies of MAC.

Acknowledgment.

This work is supported by grants from the National Science and Engineering Research Council (NSERC) and Canadian Institute for Health Research (CIHR).