Background
Helicobacter pylori is a highly host adapted bacterial pathogen, that establishes a chronic infection in the human stomach. Helicobacter pylori is transmitted orally causing a wide spectrum of diseases ranging from gastritis to gastric cancers including gastro-duodenal ulcers. The bacterium harbors a 40-kb pathogenicity island (PAI) which is associated with severe diseases.
The complete sequence of the Helicobacter pylori was determined on two isolate strains; 26695 and J99, by the Institute For Genomic Sequencing Research/Tiger and by Genome Therapeutics/Astra, respectively. Comparison of the two genome sequences shows that most genes are highly conserved and only 6 % of the genes in each genome are unique.
Construction of H. pylori MicroArray
The H. pylori genome contains 1621 putative open reading frames (ORFs). 3242 primers were designed and synthesized to amplify the complete ORFs in the H. pylori genome of strain 26695. The primers were designed to amplify each ORF beginning at the start codon and ending at the stop codon. We include in the MicroArray the super set of ORFs from both published genomes. We arbitrary chose 26695 as a reference genome and we incorporate into all forward and reverse specific primer, universal sequences of 9 bases containing uracil residues suitable for high-throughput cloning of PCR products following the Uracil DNA Glycosylase cloning method.
The PCR reactions are performed using 96-well plates in a 100 µl reaction volume. To control the yield and the specificity of the amplified ORFs, all PCR reactions were analysed by electrophoresis on a 1 % agarose gel. H. pylori MicroArray content (membranes and glass slides).
H. pylori MacroArray on nylon membrane
The 1578 PCR products that were successfully obtained (97 %) were spotted in duplicate onto positively charged nylon membrane of 12 x 8 cm and onto standard microscopic glass slides in area of 1.8 x 1.8 cm.
As hybridization signal control we include genomic DNA of strain 26695 in the DNA MicroArray spotted in duplicate. The genomic DNA was fragmented by sonication to an average length of 500 bp.
The H. pylori nylon membrane is ready to use for hybridization. After spotting, the nucleic acid samples were denatured to provide a suitable single-stranded target molecule for subsequent hybridization. Finally the H. pylori DNA MicroArray were neutralized and the samples were fixed by UV crosslinking. Quality control A- Nylon membrane Each probe was spotted onto nylon membrane in duplicate giving elements of 0.7 mm. To assess the consistency of spotting we hybridized oligonucleotides complementary to the bases of tag sequence. The amount of DNA spotted varied by 5 % between duplicate spots and 10 % between duplicate arrays. This consistency means that it is possible to compare data between different arrays.
H. pylori MicroArray on glass slides
The H. pylori 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 either better binding of the probe to the slides and accessibility to the cDNA targets to the probes.
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 H. pylori 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 pattern of spotting generates 16 blocks, each containing 18 x 17 dots with 200 microns spacing center to center
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.