We offer two N. meningitidis MicroArrays
The first one is designed for Comparative Genomic Hybridization (CGH) application to look for genomic gains and losses and to search for genetic islands which distinguish the meningococcus from the gonococcus and commensal Neisseria species (Perrin et al INFECTION AND IMMUNITY, Dec. 2002, p. 7063–7072
The second one is designed to look for changes in gene expression levels and suitable for gene profiling experiments
Construction of CGH MicroArray
The strain on which the fabrication of the CGH MicroArrays was based was N. meningitidis Z2491, a serogroup A subtype IV-1 meningococcus (sequence type ST4) isolated from an African epidemic.
Production of DNA MicroArrays Primers for PCR were designed according to the chromosomal sequence of N. meningitidis Z2491 (http://www.sanger.ac.uk/Projects/N_meningitidis/), published on the World Wide Web before annotation,to amplify contiguous stretches of DNA about 1 kb long covering the chromosome.
In order to represent all the chromosomal and have linear representation of the Z2491 genome, we walked along the chromosome and every 1 Kb we designed pairs of primers covering the entire chromosome with an overlapping of 10 to 100 bp.
The MicroArray contains up to 2073 genomic fragments (99 %) spotted in duplicate.
Construction of N. meningitidis gene expression MicroArray
The N. Meningitidis MicroArray was developed in collaboration with Dr. Xavier Nassif(Faculte´de Medecine Necker-Enfants Malades, France). Primers were designed according to the chromosomal sequence of strain Z2491 (http:// www.sanger.ac.uk/Projects/N_meningitidis/) to amplify predicted ORFs. In the case of ORFs of over 0,75 kb, only the last 0,75 kb (39 end) of the gene was amplified. ORFs present in the MC58 genomic sequence (Tettelin et al., 2000), but absent (having less than 10% predicted amino acid homology) from strain Z2491, We added to the 5' of each specific primer non-variable sequence, which is 15 bases long. This strategy allows us to perform amplification of all 2191 ORFs followed by re-amplification using single pair of universal primer. The two-step methods of amplification were designed to reduce carry-over of genomic DNA and to normalize the amount of PCR products. The main advantage of the two-step method is to introduce C6-NH2 modification to the entire PCR products necessary for covalent coupling to solid support.
All the PCR reactions were controlled by electrophoresis on agarose gel for yield and size specificity. The 5'-aminated PCR products were purified using Millipore 96-well purification kit, concentrated to 200 to 500 ng per microliter and then spotted in duplicate onto aldehyde coated glass slides.
Spotting design
The
N. meningitidis 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
N. meningitidis 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
MicroArray content
- The 98 % of N. meningitidis ORFs represented by PCR products with average size fragments of between 300 to 800 bp with a few exceptions.
- Each probe was spotted onto glass slides in duplicate giving elements of 100 microns.
- 16S ribosomal gene. Serial dilution of both Z2491 and MC58 DNA genomic.
- Luciferase and Kanamycin genes are present in each subgrid.
Spiking in known amounts of luciferase RNA showed that the arrays could consistently detect RNA at the equivalent of 10 copies per cell at levels 10 times background.
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.