Instituto de Química da Universidade de São Paulo (IQ-USP)
Manufacture protocol
A 4608-spot DNA microarray was printed containing unique internal fragments of 2692 CDS representing 94.5% of all of the 2848 CDS annotated by Simpson et al. (2000, Nature) (http://aeg.lbi.ic.unicamp.br/xf).
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 for the complete CDS list, automatically testing many parameter settings and also guaranteeing that primers hybridized only to a single genome location. A full list of primers, PCR product sizes, and their nucleotide sequences are available at the project site (http://verjo19.iq.usp.br/xylella/microarray/).
Oligonucleotides were synthesized by MWG and Operon Technologies. Genomic or cosmid DNA, obtained in the X. fastidiosa genome sequencing project, 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 µM, in a total volume of 100 µl. A 5min denaturing step at 95°C was applied, followed by 40 cycles of 95°C for 45s, 50°C for 30s, 72°C for 1min and a final step at 72°C for 10min. 4 µl 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/µl final concentration).
Generation III DNA spotter (Amersham Biosciences) was used to array the samples onto coated type-7 glass slides (Amersham Biosciences). This spotter arrays two technical replicas of each sample, one in each longitudinal half of the slides. However, this platform describes only one side of the replicated sides.
After deposition, the spotted DNA samples were crosslinked to the coated slides by applying 50 mJ of UV light and the slides were stored desiccated at ~10% relative humidity at room temperature until use.
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