The acquisition and analysis of sequence data is not an end in itself; rather it is a powerful tool to help understand the complex biology of host-pathogen interactions. It can, as Dr Barbara Gledhill finds out, be used to elucidate gene expression patterns and the relationship between genes by comparative genomics andgene product function on a whole genomic scale.
Genome sequencing must be one of if not the most exciting and dynamic areas of molecular biology. The Wellcome Trust, through its Beowulf Genomics Initiative, has dedicated at least £8m to the sequencing of genomes from bacteria and, currently, complete data have been reported for 20 prokaryotic organisms and one eukaryotic one, with another 45 and 17 respectively genomes being almost completed in laboratories around the world.
Whole genomic expression monitoring through microarray hybridisation provides a powerful approach to achieving these goals and will result in definition of differentially expressed genes important in pathogenesis. It will also provide useful targets for rational design of new drugs and vaccine candidates for bacterial pathogens. But as even small genome organisms like bacteria have around 4 x 106bp compared with 3 x 109bp in the human genome-mapping and sequencing have only become feasible through the commercial availability, over the last few years, of perfectly synthesised oligonucleotides and the total automation of the DNA sequencing process.
In the 10 years since it was founded, MWG-Biotech has rapidly established itself as a major contributor to this branch of research and in addition to an oligonucleotide synthesis service, it now offers high performance, robotic technology to handle automated pipetting, cycle sequencing and barcoded comb loading and gel loading systems that enable optimal results to be achieved within a short time.
The company is currently participating in several large Genome Sequencing Projects, and collaborating with many research groups such as that of Joseph Mangan and Philip Butcher in the Department of Medical Microbiology, St George's Hospital Medical School, London, the MRC/Wellcome Trust funded laboratory that pioneered bacterial DNA microarray technology in the UK.
DNA microarrays are invaluable for rapid and parallel analysis of the gene expression on the level of the entire genome. In this procedure, PCR products of as many genes of the organism as possible are individually immobilised on a surface to form a microscopic array. For parallel analysis of the expression profiles of all genes, multiplex hybridisation with a fluorescently labelled cDNA probe is used which is derived from the mRNA of interest for the biological question being asked by the researcher. Subsequently, the hybridisation profile is analysed by confocal microscopy using a fluorescence scanner.
Gene chip technology was pioneered by the US company, Affymetrix, which was the first to provide efficient access to genetic information using miniaturised, high-density arrays of oligonucleotide probes synthesised in situ on the surface of silicon wafers. The chips were constructed using a series of photolithographic masks to define chip exposure sites, followed by specific chemical synthesis steps.
The downside of Affymetrix's technology, however, is that not only that is it expensive but it is only available through commercial and restrictive access agreements.
Not surprisingly, the main customers are major drug companies who are using the chips for studying cancer and HIV. Indeed, chip technology may have remained beyond the means of most academic research laboratories had not Patrick O Brown of the Brown Laboratory, Stanford University Department of Biochemistry devised a method for robotic spotting of DNA onto glass slides. Robotic DNA microarraying produces lower array densities (3000cm2) than gene chips, but is more than adequate for small genome organisms like bacteria that only have between 10004000 genes.
Dr Mangan's laboratory is the first to use robotic DNA microarraying technology for bacteria in the UK which he introduced about a year ago when the first robotic instruments came on the market. In collaboration with Professor Wren of the London School of Hygiene and Tropical Medicine, microarrays are being used to study Campylobacter jejuni, the major food poisoning bacterium in this country. A second major focus of the St George's lab is on the TB bacterium M tuberculosis.
According to Dr Mangan, the TB bacterium is the biggest killing single micro-organism in this world, with 10 million deaths and three million new cases p.a.One third of the world's population are latently infected with TB. The bacterium may be lying dormant and the individuals showing no physical symptoms but were it to activate, we currently have no treatment other than surgery. The increasing incidence of TB and the impact of HIV and the emergence of multiple drug resistance has led the World Health Organisation to declare TB a global emergency.
Gene expression profile
With the whole genome of M tuberculosis now sequenced at the Sanger Centre (Cole et al Nature 1998), Dr Mangan is studying its gene expression profile; looking at the expression of TB genes when cultured in vitro compared with when TB is inside macrophages or during models of infection. He extracts total RNA from macrophage-derived TBs and from TB in vitro and compares the gene expression profile in both to see what the differences are so that these can be targeted for further study.Genes expressed in macrophages are likely to be of interest as targets for a new generation of antibiotics and better vaccines.
To produce DNA microarrays on glass microscope slides, Dr Mangan uses a MWG-Biotech RoboAmp 4200 pipetting robot with integrated Primus HTR thermocycler, a gridding robot and a confocal laser scanner/gridder configured to read the slides.
The first stage is to make PCR primers of all the 3924 genes in the TB genome, and for this Dr Mangan uses the MGW RoboAmp 4200 with oligo nucleotides synthesised by MWG Biotech (Fig. 1). The PCR primers are all tested by electrophoresis for integrity, and the PCR products are then robotically spotted onto microscope slides. The slides are hybridised with fluorescently labelled DNA or cDNA using two different dyes to compare the two conditions and a confocal laser scanner is used to pick up the colour fluorescence.
The 4000 PCRs take a week to make with the RoboAmp 4200 robot; and the gridding robot takes about 3 hours to place the 4000 genes on each of 20 microscope slides for scanning.
The instrument is also novel in that it uses carbon coated conductive tips. The big advantage of these is that the robot always knows that the tip is there, which is not necessarily the case with conventional clear plastic pipette tips. It is also possible to set the robot to track just underneath (2mm) the meniscus of the liquid being pipetted so it tracks down the lowering column of liquid. This minimal pipette tip contact is again a boom in preventing cross contamination.
Under computer control, the micoplates are moved automatically from storage to pipetting positions into the thermocycler or onto the stacker. The robot also loads aliquots of the PCR primers onto electrophoresis gels which is done both to check that the PCR has worked and that the bands are of the right size.The rest of the PCR products are then aliquotted from four 96 well plates into one 384 well plate, ready for the gridder robot to transfer them to glass microscope slides for assaying.
Spotting takes place via a BioRobotics Microgrid which uses a pin tool to spot the 4000 TB PCRs onto a single microscope slide. Quill pin modules are becoming popular because they can multidispense spot from one loading; the quill pin module picks up from 16 wells of the 384 well plate at a time, and spots them onto 50 slides. The quills are then washed in water, ethanol and dried, ready to pick up from the next 16 wells.
After spotting is finished, the glass microscope slides are hybridised with cDNA labelled with either Cy3 (red) or Cy5 (green) dyes depending upon whether the mRNA is from macrophage-derived TB or in vitro grown TB. The slides are then read by a ScanArray 3000 Confocal laser scanner.
There are two lasers in the scanner. One is tuned to the excitation level of Cy3 and the other to Cy5. If images are overlayed, genes only expressed inside macrophages will appear as red spots. If they are only in vitro, they will appear as green spots. If in both, yellow/brown spots appear indicating coexpression.
