Streptococcus pneumoniae MicroArray was designed and developed in close collaboration with the Molecular Genetics group at the University of Groningen, The Netherlands (Prof. Dr. O.P. Kuipers, Dr. J. Kok) and Laboratory of Pediatrics, Erasmus Medical Center, Rotterdam (Dr. P.W.M. Hermans, Prof. Dr. R. de Groot). The genome sequence of the gram-positive pathogenic bacterium Streptococcus pneumoniae has been released and the sequence information can be obtained at ftp://ftp.tigr.org/pub/data/s_pneumoniae/. From 2105 unique sequences provided on the web site we select 2085 ORFs. Specific primers were designed to amplify all 2085 putative ORFs. The primers were selected carefully to amplify only a specific region of each ORF. Universal sequences of 15 bases were incorporated on the 5' of each specific forward and reverse primer. The PCR reactions were performed in two rounds using 96-well plates in a 100 ml reaction volume. The first round of PCR reaction (primary PCR) was done using 10 ng of DNA genomic as template and the specific part of the oligo. The second round of PCR reactions (secondary PCR) was performed in the presence of 5 ul of 40-fold diluted primary PCR products and the C12-NH2 modified universal primers. The entire primary and the secondary PCR reactions were controlled on standard gel electrophoresis for yield and for the expected size. Using these two-step methods we have successfully amplified up to 96 % of 2085 S. pneumoniae ORFs. The 5'-aminated PCR products were purified using Millipore 96-well purification kit, concentrated to 200 to 500 ng per microliter and then covalently immobilization onto aldehyde coated glass slide through a well-known Schiff base reaction. S. pneumoniae MicroArrays are manufactured by printing PCR amplicons suspended in optimized spotting buffer for high coupling efficiency of DNA to the most consistent aldehyde coated glass slides. This spotting chemistry provides us the highest hybridization intensities, which is a consequence of a better binding of the probe to the slides and their accessibility to the cDNA targets. We use ChipWriter™ Pro (Virtek) which is a high precision dispensing robot to spot the PCR products onto aldehyde glass slides. The robot is designed to collect 100 nl of DNA solution and to deposit 0.6 nl per spot. For S. pneumoniae MicroArray, we use simultaneously 16 pins to place the spots in area of 1.8 x 1.8 cm. In order to generate high quality spots, the printing procedure is performed under a tight controlled humidity and temperature environment. This allows having the complete control on the spot morphology, the spot diameter and uniformity. This configuration of the robot generates 16 blocks, each containing 15 x 15 dots with 200 microns spacing center to center with a spot diameter of 100 microns. Note that the pattern of spotting could be different from the one mentioned above.
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