PCR primers (20-mers) were designed to amplify the full-length coding DNA segments of 3080 predicted ORFs based on the annotation of the complete C. glutamicum R genome sequence (NC_009342). Amino-terminus primers amplified 20 bp downstream from the initiation codon, which was included. Carboxy-terminus primers amplified 20 bp upstream from the termination codon, which also was included. The ORF-specific double-stranded DNA fragments were produced by standard PCR amplification methods, as described above, with genomic DNA of C. glutamicum R as template and the above primer sets. Each PCR product was analysed routinely by agarose gel electrophoresis to ensure its size and purity. When PCR products of the expected size or purity were not obtained, modification of PCR conditions, redesign of primer pairs, or purification of DNA fragments from gels were attempted. As a result, a library of 3076 single PCR products was obtained that contained 99.9 % of a total of 3080 predicted ORFs present in the C. glutamicum R genome. PCR products were purified by a PCR96 Cleanup kit (Millipore), desiccated and resuspended in Solution-T (Takara Bio). The PCR products thus obtained were printed in duplicate at two separate positions onto a 1x3 inch (2.5x7.5 cm approx.) Takara slide glass TX704 (Takara Bio) using an arraying robot. The DNA microarrays were rehydrated in a humidity chamber at 37 °C for 1 h. They were subsequently exposed to UV light at an energy setting of 60 mJ cm–2 in a UV cross-linker, and soaked in 0.2 % SDS for 2 min at room temperature. After being washed twice with ultrapure water, the slides were immersed in a succinic anhydride solution for 20 min at room temperature, and placed in boiling water for 2 min. The slides were then immersed in 100 % ethanol for 1 min and dried by centrifugation at 185 g for 2 min. Spotting quality was assessed by checking as controls the configuration and concentration of non-specific stained DNA spots on the DNA microarrays. In addition, 12 spots each of human transferrin receptor (1 kb), E. coli plasmid pUC19 and {lambda}DNA were printed onto the slides as negative controls for DNA hybridization.
Less...
More...