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10 analysis and control Microarrays and genomics bring new insights to the food industry IFR (the Institute of Food Research) is the leading centre in Europe for its work with Salmonella microarrays, and in this article Dr Jay Hinton will use the Institute’s experiences with Salmonella to explain how microarray technology works, and what it can be used for. The first genome sequence for Salmonella was completed in 2001, and gives a complete readout for every gene in the organism. It described the 5000 separate genes of Salmonella typhimurium, one of the major causes of food-poisoning in humans in Europe. Since 2001 five new genome sequences have been completed for Salmonella, including Salmonella enteritidis, another major cause of food poisoning associated with chicken eggs. The scope of the problem is significant: in the UK alone there are at least 50,000 cases of foodborne Salmonella infection every year and 100 deaths. It kills more people than any other bacterium that is transmitted by food. The major culprits are poultry, pork and eggs, but it can also be transmitted on vegetables such as salads. Those most at risk will be people who are immunocompromised, particularly the very young or the very old, which is why eggs used in residential homes for the elderly are pasteurised. Developing the microarray DNA Microarrays are new tools that allow researchers to learn the function of newly-identified genes from bacterial genomes. The concept of microarray technology was first developed in 1995 in Stanford, California, by two scientists called Pat Brown and Joe DeRisi. It was a completely original idea, made possible by various different technical developments, particularly the development of accurate robots to make silicon chips. In simple terms, a microarray is a small, glass microscope slide, and the surface is covered with thousands of DNA spots representing different genes. Once the genome sequence for Salmonella typhimurium had been completed, the team at IFR started to develop a microarray. They used a process known as PCR (Polymerase Chain Reaction) to amplify the DNA, make copies of every Salmonella gene and put a copy of each gene as a spot onto glass slides. Main challenges in the development phase However, the development of the microarray was not completely straightforward. The first task was to get the robotics side of things working. Because there were problems with many of the commercial offerings, the team at IFR ended up building their own robotic printer to make the microarrays. That required some technical know-how, but fortunately the group in Stanford had made the plans for their robot available on the Internet, enabling food spring 2005
analysis and control 11 IFR to buy the parts from America and build it. This was a relatively easy process and in 2001 the group even published a paper entitled ‘It’s easy to make your own microarrayer’. They encouraged others to do the same, but most biologists don’t feel confident in the field of electrical engineering! One project is to ask what happens during the lag phase of bacterial growth. This is the earliest stage of growth Having made the machine, the next challenge was to get it working. That was not difficult in terms of the machinery, but it was hard to optimises the hybridisation process – mixing fluorescentlylabelled DNA with the microarray, and at this point the team visited Stanford for advice. Success with this technology very much requires laboratories to work together and not in isolation. Genomic indexing There are two main applications for the microarray. Firstly, it can be used to examine the DNA in bacteria, and to generate the world’s most detailed fingerprint that exactly describes which of the 5000 Salmonella genes are present . If you’re interested in typing bacteria or looking to see if bacteria contain genes that would allow them to be virulent or infectious, you can grow the bacteria in the laboratory, isolate the chromosomal and plasmid DNA, and then fluorescently label the DNA red or green and mix it onto the microarray. If a gene is one of the 5000 spots and is present in the bacteria, it will light up with a fluorescent dye. This is essentially a plus/minus process known as genomic indexing. With the newly discovered genes for the different strains of Salmonella also present on the microarray, you can take any Salmonella from the environment, isolate the DNA, put it onto the microarray and look at which genes are present/absent. In practical terms, if there is an outbreak of Salmonella poisoning and you want to determine if it is linked to your factory, this approach can be used to type the Salmonella from the outbreak and to ask if is it present. Conversely, if you find Salmonella in your factory and wish to discover whether or not it is particularly dangerous, you can put the genomic DNA onto the microarray and see whether the virulence genes, required for infection, are present. If not, then it is unlikely to cause infection. In other words, the microarray has the potential to assess how hazardous the bacterium is. Other developments There are other methods of bacterial typing, many of which are done in microbiology laboratories involving biochemical methods and phage typing. Alone, these are somewhat limited, and microarray technology complements them. While microarray technology is still based in the laboratory, there are some opportunities on the horizon. For example, scientists are working on developing an assay that will tell you in a couple of It is well known that acid can be used to kill bacteria, as can temperature, but we don’t understand much about the mechanism – the research has been somewhat empirical hours whether hundreds of important genes are present or absent in bacteria. This work is being done at the Veterinary Laboratories Agency, and is intended to develop a system that will have no need for a fluorescent scanner. When that has been developed, perhaps in 3–4 years, the technology may well find itself being used in smaller businesses. Switching genes on or off Asking whether genes are present or absent is relatively easy. It is more difficult to ask whether a gene is being switched on or off. When a gene is switched on it makes RNA and then protein which allows it to carry out certain activities. It might a protein that allows the bacterium to survive in an acid environment or a component needed to make a cell wall. By looking at the RNA (transcriptomics), you can get a readout of all the genes that have been switched on or off in different environments. Happily, the chemical nature of RNA allows one to be confident that you are working with a complete set of all the RNA of the bacterium, which is much easier than trying to work with all of the proteins. Proteomics is technically more challenging because it is difficult to isolate all proteins and render them soluble. In Salmonella infection, for example, you may eat a bacterium on a cold lettuce leaf. Once it gets into the mouth it becomes warm and switches on genes to react to that temperature stress, then it passes into the stomach where it is suddenly plunged into an acid bath and swiftly has to turn on genes that allow it to survive the low pH environment. From there it enters the small intestine, where it’s salty and no oxygen is present. This forces it to switch on yet more genes as well as genes that allow it to use the different carbon sources that are available in the gut. Microarray tools are ideal for examining that adaptability and for using the bacteria as a ‘sensor’, because the readout from the microarray describes what the bacteria are doing For example, food spring 2005
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