Development and validation of Corynebacterium DNA microarrays

We have developed DNA microarray techniques for studying Corynebacterium gl utamicum. A set of 52 C, glutamicum genes encoding enzymes from primary met abolism was amplified by PCR and printed in triplicate onto glass slides. T otal RNA was extracted from cells harvested during the exponential-growth a nd lysine production phases of a C. glutamicum fermentation. Fluorescently labeled CDNAs were prepared by reverse transcription using random hexamer p rimers and hybridized to the microarrays, To establish a set of benchmark m etrics for this technique, we compared the variability between replicate sp ots on the same slide, between slides hybridized with cDNAs from the same l abeling reaction, and between slides hybridized viith cDNAs prepared in sep arate labeling reactions. We found that the results were both robust and st atistically reproducible. Spot-to-spot variability was 3.8% between replica te spots on a given slide, 5.0% between spots on separate slides (though hy bridized with identical, labeled cDNA), and 8.1% between spots from separat e slides hybridized with samples from separate reverse transcription reacti ons yielding an average spot to spot variability of 7.1% across all conditi ons. Furthermore, when we examined the changes in gene expression that occu rred between the two phases of the fermentation, we found that results for the majority of the genes agreed with observations made using other methods , These procedures will be a valuable addition to the metabolic engineering toolbox for the improvement of C, glutamicum amino acid-producing strains.