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Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome

DOI: 10.1186/1756-0500-3-150

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The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration.Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas.Citrus canker, or cancrosis, is a disease that affects most species of the Citrus genus [1]. Its symptoms can be observed on leaves, fruits or branches and are characterized by small, pointed and spongy pustules that are surrounded by a chlorotic halo on both sides of the leaf. This halo tends to spread quickly through the tissue, increasing its diameter and becoming brownish or darker in color with a rough and salient appearance [2]. The causal agent of cancrosis is the bacterium Xanthomonas axonopodis pv. citri (XAC) [3], more recently renamed as Xanthomonas citri subsp. citri [4,5] whose genome was completely sequenced in 2002 [6] and compared with others organisms [6-8]. Data from this sequencing project revealed that the XAC genome contains a circular chromosome of 5.2 Mbp and two plasmids (33 and 64 Kbp), containing a tota

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