DNA Microarray Construction. PCR primers were designed to amplify unique internal fragments of 200-1000 bp of each predicted CDS described in the annotated genome sequence of X. fastidiosa strain 9a5c (http://aeg.lbi.ic.unicamp.br/xf). Primers (18-23mers) with equivalent predicted melting temperature were designed with the use of a perl program that ran PRIMER3 (http://www-genome.wi.mit.edu/genome_software/other/primer3.html) for the complete CDS list, automatically testing many parameter settings and also guaranteeing that primers hybridized only to a single genome location. Oligonucleotides were synthesized by MWG and Operon Technologies. Genomic or cosmid DNA, obtained in the X. fastidiosa genome sequencing project (Simpson et al. 2000), were used as template in the first round of PCR amplification, and 200-fold-diluted PCR products were used as templates for PCR reamplification to increase product concentration when necessary. The reactions were done in 96-well plates. The mixture in each well contained 100 ng of DNA, 0.5 U of Biolase Taq polymerase (Bioline), 0.2 mM of each dNTP (Invitrogen), 1.5 mM MgCl2 and the primers at 0.5 uM, in a total volume of 100 ul. A 5min denaturing step at 95oC was applied, followed by 40 cycles of 95oC for 45s, 50oC for 30s, 72oC for 1min and a final step at 72oC for 10min. 4 ul of each PCR reaction were checked for product size and concentration by electrophoresis in 1.2% agarose gels. The amplicons were then purified with 96-well MultiScreen purification plates (Millipore) and an equal volume of dimethyl sulfoxide was added to the purified products (~100 ng/ul final concentration). Generation III DNA spotter (Amersham Biosciences) was used to array the samples onto coated type-7 glass slides (Amersham Biosciences). After deposition, the probes were crosslinked to the slides by applying 50 mJ of UV light and the slides were stored desiccated at ~10% relative humidity at room temperature until use. Keywords = xylella fastidiosa Keywords = genomotyping Keywords = DNA microarray Keywords = comparative genomics
