|Kapur, Vivek - UNIV OF MN|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: September 18, 2003
Publication Date: N/A
Technical Abstract: Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease, a chronic infectious ailment of ruminants that results in severe economic loss to the dairy industry. Despite previous studies aimed at elucidating the pathogenesis of M. paratuberculosis, nothing is known about the type of transcriptional response utilized upon the infection of a host animal. Using the recently completed genome sequence of M. paratuberculosis, we have begun to construct a DNA microarray representing all of the identified open reading frames (ORFs). A library of PCR products was constructed with primers designed to amplify fragments £ 500 bp from each ORF using purified genomic DNA as a template. Two successive rounds of PCR were performed to minimize genomic DNA contamination in the final amplified products. The final 50 mL reactions were checked for quality on agarose gels and purified with filter plates. Initially, greater than 80% of the M. paratuberculosis ORFs were successfully amplified. Additional attempts are needed to amplify the remaining ORFs. PCR products will then be arrayed in triplicate onto poly-L-lysine coated slides using a precision arraying robot. Three Arabadopsis genes will be included on the array for controls. The finished arrays will be valuable in future studies examining the transcriptional response of M. paratuberculosis to a variety of environmental stimuli.