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Download Microarrays = Gene Chips
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Microarrays = Gene Chips What is a Microarray (Gene chip)? • A microarray is an orderly arrangement of short pieces of single stranded (unpaired) DNA in a grid on a suitable surface - often a glass slide • A specialized robot places the spots at precise intervals (20 to 100 mm diameter) • Each spot is a single sequence (gene) • Each spot contains millions of copies of that single sequence – A bacterium’s entire genetic make-up can be contained on a single gene chip • A computer keeps track of which piece of DNA is contained in each spot • Tens of thousands of spots on a single slide How does it work? • Single stranded DNA likes to form a double stranded molecule by matching with a complementary strand • One of the basic driving properties of DNA • Microarrays or gene chips take advantage of this – DNA:DNA or DNA:RNA • Based on the ability of single strands of DNA to hybridise (stick) to complementary (matching) sequences • Example, a scientist might wish to figure out which bacteria are present in a soil sample Process: DNA:DNA 1. Buy a gene chip with genes representative of bacterial species (dictionary of barcodes) 2. Extract DNA from the soil sample 3. Use PCR to amplify genes that identify different bacteria (barcodes) Extract DNA 4. Label the PCR products with a fluorescent dye PCR 5. Wash the labelled PCR products over the gene chip and allow complementary strands to stick together 6. Wash off any unstuck PCR products 7. Use a laser to detect the fluorescent dyes and create a visual image of the pattern of the dyes 8. If the PCR product has stuck on it will glow 9. The computer can then say which of the bacterial species the PCR products have stuck to and this indicates which species are present in the sample + Label Process: DNA:RNA • Question – what genes are active in the “on state” • Looking at all the genes expressed in a cell, some are specific but some are general (eg those involved in respiration) OFF • Extract mRNA from two cells – one in off state and one in on state • Label one green and one red • Wash over a gene chip that has a selection of genes on it • If the gene matches it will bind to the chip • Excite with two lasers – one for the green dye and one for the red dye – computer overlays the image Stick to microarray ON • Green = only in off state • Red= only in on state • Yellow = equal in both states www.brunel.ac.uk/.../researchgroups/mf/mp 'GreeneChip' - New diagnostic tool that rapidly and accurately identifies multiple pathogens • • • • • • • The Greenechip System Diagnosis of infectious diseases, including those caused by viruses, bacteria, fungi, or parasites Can be used on a wide variety of samples, including tissue, blood, urine, and stool Can get an answer in 6 hours The chip has almost 30,000 pieces of genetic material taken from thousands of different viruses, bacteria, fungi and parasites – represent all recognized 1,710 vertebrate viral species and 135 bacterial, 73 fungal, and 63 parasite genera. For each family or genus at least 3 separate genomic target regions are used The test was used on a sample from a healthcare worker who had died of fever and liver failure during the Marburg outbreak in Angola. Previous tests for hemorrhagic fevers had been negative. The GreeneChip result pointed to Plasmodium falciparum malaria ( www.cdc.gov/EID/13/1/06-0837.htm). Plant health diagnostics on a microchip In Europe: • A wide range of organisms can attack plants, from viruses to insects • Those that are serious pests and diseases are quarantined under the EU Plant Health Directive (almost 300 pathogens) • This legislation controls the passage of plant material between countries • To implement the legislation, Member States and Acceding countries must be able to diagnose all the pests and pathogens, some of which are obscure • Currently, diagnosis requires a wide range of expertise, from traditional taxonomy to classic microbiology and molecular biology • The Diag Chip project, with a consortium of plant disease scientists from Germany, France, Spain and the UK aimed to create a gene chip for this detection – currently under construction Example • • Previously no means to identify new cancer classes (class discovery) or for assigning tumours to known classes (class prediction) Important for targeting treatment 1999 Science 286:531-537 • The 50 genes most highly associated with the ALL-AML class distinction • row = a gene • column = a patient • Expression levels are normalised using the mean • > mean = red • < mean = blue