1. Microarry ( DNA microarray) S.Rasoulinejad Ms.c Microbial Biotechnology Prof. Mousavi

2. Microarray Introduction principle Applications Advantages Limitations References 3. introductions 4. history Microarray technology evolved from Southern blotting. The concept of microarrays was first proposed in the late 1980s by Augenlicht and his colleagues. They spotted 4000 cDNA sequences on nitrocellulose membrane and used radioactive labeling to analyze differences in gene expression patterns among different types of colon tumors in various stages of malignancy. 5. PRINCIPLE The principle of DNA microarrays lies on the hybridization between the nucleotide. Using this technology the presence of one genomic or cDNA sequence in 1,00,000 or more sequences can be screened in a single hybridization. Fluorescent labeled target sequences that bind to a probe sequence generate a signal that depends on the strength of the hybridization determined by the number of paired bases. 6. why use this technic? By using an array containing many DNA samples, scientists can determine, in a single experiment, the expression levels of hundreds or thousands of genes within a cell by measuring the amount of mRNA bound to each site on the array. 7. DNA MICROARRAY TECHNOLOGY DNA microarray technology defined as a high-throughput and versatile technology used for parallel gene expression analysis for thousands of genes of known and unknown functions. A DNA microarray is a collection of microscopic DNA spots on solid surface. Each spot contains picomoles of a specific DNA sequence, known as probes or reporters. A microarray is a pattern of ssDNA probes which are immobilized on a surface called a chip or a slide. Microarrays use hybridization to detect a specific DNA or RNA in a sample. 8. Contact Printing InkJet (HP/Canon) technology 1 drop = 100 picolitres Non Contact Printing 9. Technique used for the Preparation of DNA chip Fig: Photolithography 10. The colors of microarray. GREEN represents Control DNA, where either DNA or cDNA derived from normal tissue is hybridized to the target DNA. RED represents Sample DNA, where either DNA or cDNA is derived from diseased tissue hybridized to the target DNA. YELLOW represents a combination of Control and Sample DNA, where both hybridized equally to the target DNA. BLACK represents areas where neither the Control nor Sample DNA hybridized to the target DNA. Each spot on an array is associated with a particular gene. Each color in an array represents either healthy (control) or diseased (sample) tissue. Depending on the type of array used, the location and intensity of a color will tell us whether the gene, or mutation, is present in either the control and/or sample DNA. It will also provide an estimate of the expression level of the gene(s) in the sample and control DNA. 11. - Clustering Idea: Groups of genes that share similar function have similar expression patterns Hierarchical clustering k-means Bayesian approaches Projection techniques Principal Component Analysis Independent Component Analysis - Classification Idea: A cell can be in one of several states (Diseased vs. Healthy, Cancer X vs. Cancer Y vs. Normal) Can we train an algorithm to use the gene expression patterns to determine which state a cell is in? Support Vector Machines Decision Trees Neural Networks K-Nearest Neighbors 12. Of MicroarrayApplications GENE EXPRESSION DISEASE DIAGNOSIS DRUG DISCOVERY TOXICOLOGICAL RESEARCH 13. MICROARRAY AS A GENE EXPRESSION PROFILING TOOL The principle aim of using microarray technology as a gene expression profiling tool is to answer some of the fundamental questions in biology such as "when, where, and to what magnitude genes of interest are expressed. Microarray analysis measure changes in the multigene patterns of expression to better understand about regulatory mechanisms and broader bioactivity functions of genes. 14. Gene expression Profiling The expression levels of thousands of genes are simultaneously monitored to study the effects of certain treatments, diseases , and developmental stages on gene expression. Conditions Genes How experimental conditions influenced production (expression) of mRNA for a set of genes. Green indicates reduced expression. Cluster analysis has placed a group of down regulated genes in the upper left corner. 15. MICROARRAY AS A COMPARATIVE GENOMICS TOOL Microarray technology have widespread use in comparative gene mutation analysis to analyse genomic alterations such as sequence and single nucleotide polymorphisms. In microbiology microarray gene mutation analysis is directed to characterisation of genetic differences among microbial isolates, particularly closely related species. 16. DISEASE DIAGNOSIS Different types of cancer have been classified on the basis of the organs in which the tumors develop. Now, with the evolution of microarray technology, it will be possible for the researchers to further classify the types of cancer on the basis of the patterns of gene activity in the tumor cells. Use in diagnosis (presence) Example: characterizing acute lymphoblastic leukemia. Also breast cancer. Use in prognosis Example: assessing the likelihood of metastasis in medulloblastoma (brain tumor in children) 17. TOXICOLOGICAL RESEARCH Microarray technology provides a robust platform for the research of the impact of toxins on the cells and their passing on to the progeny. Toxicogenomics establishes correlation between responses to toxicants and the changes in the genetic profiles of the cells exposed to such toxicants. The microarray permits researchers to examine thousands of different genes in the same experiment and thus to obtain a good understanding of the relative levels of expression between different genes in an organism. 18. Advantages of DNA Microarrays. DNA microarrays are better than other profiling methods ,it has features such as : Easier to use Are high-throughput (can analyze thousands of genes or markers at a time) Generate large amounts of data in little time Do not require large-scale sequencing - Allow the quantitation of thousands of genes from many samples 19. Limitations of array. Do not necessarily reflect true levels of proteins - protein levels are regulated by translation initiation & degradation as well The production of too many results at a time requires long time for analysis, which is quite complex in nature. Expensive! $20-$100K to make your own / buy enough to get publishable data The DNA chips do not have very long shelf life, which proves to be another major disadvantage of the technology. 20. Thank you