Oligonucleotides design and synthesis
The development of Yarrowia lipolytica DNA MicroArray representing 6650 putative ORFs was carried out in collaboration of Prof. Angel Dominguez (Spain) Prof. Kim (South Korea), Prof. Stefan Kerscher (Germany), Prof. Gerold Barth (Germany) and Prof. Claude Gaillardin (France).
The total of 6650 oligonucleotides of 60 bases were designed and the synthetic oligonucleotide probes were produced at EUROGENTEC (Liège, Belgium).
The oligonucleotide were synthetized using the standard phosphoramidite chemistry. For compatibility reason with surface chemistry of dendrolames, epoxy and aldehyde coated glass slides which require C6-NH2 modification for permanent attachment of oligonucletides, we added to all the oligonucleotides the C6-NH2 linker at their 5' end.
The complete set of oligonucleotides was aliquoted into several replicate plates. An aliquot of 384-well plates from the set of oligonucleotides was hydrated in water then diluted to the printing concentration with EGT spotting solution.
Robotic Gridding onto Glass slides
Microarrays are prepared by printing amino-modified long oligos suspended in EUROGENTEC specific buffer. This buffer is optimized for different type of glass slide surface. Robotic Gridding onto Glass slides Microarrays are prepared by printing 6650 oligonucleotide suspended in our specific buffer on high quality glass slides, which offer the following key features:
- Coated with a polymer containing multifunctional reactive groups
- High DNA immobilization efficiency
- Enhanced Cy® 5 signal and stability
- Excellent spot morphology
- Stable in high humidity environment
- Excellent batch-to-batch consistency
For Microarray printing we use ChipWriterPro, which is a microarray robot built by ESII (Toronto, Canada) and distributed by Bio-Rad. For 6650 oligonucleotides, the arrayer uses 32 pins to print DNA samples from 384-well microtiter plates onto glass slides. All the oligos are spotted as a duplicate and they are placed in area of 3.2 x 1.8 cm. The MicroArray contains 32 blocks; each block contains 416 dots. The distanced between spot to spot is ranging from 90 to 120 microns with 100 microns of spot diameter. The slides can be stored at room temperature for three months without lost of signal intensities of hybridization. .
The MicroArray includes serial dilution of Luciferase gene allowing you to normalize the signal of hybridization and to evaluate the dynamic range of the scanners. For negative control we incorporate to the MicroArray three distinct oligonucleotides with 70, 50 and 30 % identity of sequence with some highly expressed Y. lipolytica genes. This allows you to evaluate the efficiency of hybridization and washing conditions.
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.