Construction of S. pombe MicroArray
We developed the
S. pombe MicroArray in collaboration with the Södertörns University College in Sweden and the Max Planck Institute in Berlin, Germany.
Up to 4976 specific primer pairs have been designed and synthesized to amplify all the putative ORFs. The MicroArray covers up to 99.5 % of the annotated fission yeast genes.
We added a universal TAG sequence of 14 and 15 bases to all forward and reverse primers respectively. The two-step method of PCR reaction generates C6-amino modified PCR products with an average length of 500 bp.
Spotting design
S. pombe 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
C. albicans MicroArray, we use simultaneously 32 pins to place the spots in area of 3.6 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 32 blocks, each containing 20 x 20 dots with 200 microns spacing center to center with a spot diameter of 100 microns.
MicroArray content
- 4976 ORFs spotted in duplicate
- Mitochondrial genes
- Tags (GFP, TAP, GST)
- Marker genes (Kan, Leu2, LacZ)
- Positive controls for spike-in RNA (luciferase)
- Negative controls (introns and intergenic regions)
- Cross-hybridization controls (S. cerevisiae genes with 70 %, 50 % and 30 %homology to the S. pombe genes).
Legal notices
The use of this Array-related product in relation to the manufacture or use of nucleic acid arrays may be covered by the following patent owned by Oxford Gene Technology Limited ("OGT"): EP 0,373,203. The purchase of this product does not confer on the purchaser any rights or licences under any OGT patent. To enquire about a licence under OGT's patents, please contact licensing@ogt.co.uk.