Construction of S. cerevisiae MicroArray
Comparative genome analysis of 6213 predicted Saccharomyces cerevisiae open reading frame (ORF) products with 13 yeast genome sequence related species proposed the elimination of 612 predicted ORFs that are not real genes (A. Malpertuy et al, FEBS Letters, Volume 487, 22 December 2000).
The study identified the presence of 50 new genes. Specific pair of primers was designed by Pasteur Institute, Paris to amplify all 5803 ORFs non-spurious coding sequences.
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 two-step PCR method has been employed to generate 5' amino-modified PCR for covalent attachment to the aldehyde-coated support. All the amplicons have an average length of 300 bp and they were controlled systematically by electrophoresis on agarose gels.
Spotting design
The yeast 5803 ORFs MicroArray is composed of 32 subgrids, which follow a pattern of 8 metarows and 4 metacolumns. Each subgrid contains 20 columns and 21 rows with a dot spacing of 200 µm. Probes are spotted as closed duplicates.
In order to provide a friendly environment, the first subgrid row on each array metarow contains all the normalization controls, which include the controls with serial dilution of the Renilla LuxA cDNA PCR product.
In addition to these controls, four serial dilutions of the sonicated yeast S288c genomic DNA are also located in the corner subgrids of the MicroArray.
MicroArray content
To determine the background values, the stringency of the hybridization and washing conditions we add the following probes to the MicroArray:
1- Three
E. coli genes showing no homology with yeast ORFs (aceF, kdtA and tufA)
2- Ten yeast intergenic regions 3- Three orthologous genes from Schizosaccharomyces pombe Leu1, His1 and Ura4 and printing buffer are spotted on the MicroArray (negative controls).
3- Marker genes are also present into the MicroArray in order to follow the expression of genes of interest (Schistosoma mansoni 28 kDa glutathione S-transferase, Aequorea victoria green-fluorescent protein GFP, TAP fusion tag, human c-myc tag, LexA DNA binding domain , Pho4 DNA binding domain, b-galactosidase LacZ 5'- and 3'- encoding regions) or the genetic background of a KanMX-modified strain.
Finally, two sets of oligonucleotides cover the large genes YKL182w (6.153 kbp, 11 oligonucleotides), and YLR310c (4.567 kbp, 9 oligonucleotides) to allow the user to monitor the efficiency of the reverse transcription reaction.
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.