Analysis of gene expression

Analysis of gene expression

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  AnAlysis of Gene expression AnAlysisof Gene expression Tapeshwar Yadav (Lecturer) BMLT, DNHE, M.Sc. Medical Biochemistry
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  AnAlysis of Geneexpression The tools of biotechnology not only allow the study of gene structure, but also provide ways of analyzing the products of gene expression - mRNA and proteins A.Determination of mRNA levels B.Analysis of proteins
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  A. DeterminAtion ofmrnA levels Messenger RNA levels are usually determined by the hybridization of labelled probes to either mRNA itself or to cDNA produced from mRNA. 1.Northern blots: 2.Microarrays:
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  1.northern blots: Northern blotsare very similar to Southern blots except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then transferred to a membrane and hybridized to a radioactive probe. The bands obtained by autoradiography give a measure of the amount and size of particular mRNA molecules in the sample.
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  2. Microarrays: DNA microarrayscontain thousands of immobilized DNA sequences organized in an area no larger than a microscope slide.  These microarrays are used to analyze a sample for the presence of gene variations or mutations (genotyping), or to determine the patterns of mRNA production (gene expression analysis), analyzing thousands of genes at the same time. For genotyping analysis, the cellular sample is genomic DNA. For expression analysis, the population of mRNA molecules from a particular cell type is converted to cDNA and labelled with a fluorescent tag.
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  Contd…  This mixtureis then exposed to a gene chip, which is a glass slide or membrane containing thousands of tiny spots of DNA, each corresponding to a different gene.  The amount of fluorescence bound to each spot is a measure of the amount of that particular mRNA in the sample.  DNA microarrays are often used to determine the differing patterns of gene expression in two different types of cell—for example, normal and cancer cells. [Note: Physicians hope to one day be able to tailor particular treatment regimens to each cancer patient, based on the specific microarray expression patterns exhibited by that patient's individual tumor.]
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  B. AnAlysis ofproteins The kinds and amounts of proteins in cells do not always directly correspond to the amounts of mRNA present. Some mRNA are translated more efficiently than others, and some proteins undergo posttranslational modifications by adding sugars or lipids, or both.  Thus, the genome contains 20,00 to 30,000 genes, but a typical cell produces hundreds of thousands of distinct proteins.
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  Contd… When investigating one,or a limited number of gene products, it is convenient to use labelled antibodies to detect and quantify specific proteins.  However, when analyzing the abundance and interactions of large numbers of cellular proteins (called proteomics), automated methods employing two-dimensional gel electrophoresis, mass spectrometry, multidimensional liquid chromatography, and bioinformatics are employed
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  1.enzyme-linked immunosorBent AssAys (elisA): Theseassays are performed in the wells of a plastic microtiter dish. The antigen (protein) is bound to the plastic of the dish. The probe used consists of an antibody specific for the particular protein to be measured. The antibody is covalently bound to an enzyme, which will produce a colored product when exposed to its substrate. The amount of color produced can be used to determine the amount of protein (or antibody) in the sample to be tested.
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  2. Western Blots: Westernblots (also called immunoblots) are similar to Southern blots, except that protein molecules in the sample are separated by electrophoresis and blotted (transferred) to a membrane. The probe is a labeled antibody, which produces a band at the location of its antigen.
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  3. Detecting exposureto HiV:  ELISA and Western blots are commonly used to detect exposure to HIV by measuring the amount of anti-HIV antibodies present in a patient's blood sample.  ELISA are used as the primary screening tool, because they are very sensitive.  These assays sometimes give false positives, however, so Western blots, which are more specific, are often used as a confirmatory test . [Note: ELISA and Western blots can only detect HIV exposure after anti-HIV antibodies appear in the bloodstream. PCR-based testing for HIV is more useful in the first few months after exposure.]
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  4. proteomics: Involves thestudy of all proteins expressed by a genome, including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other macromolecules. The 20,000 to 30,000 genes of the human genome translate into hundreds of thousands of proteins when alternate splicing and posttranslational modifications are considered. While a genome remains unchanged, the amounts and types of proteins in any particular cell change dramatically as genes are turned on and off. Proteomics offers the potential of identifying new disease markers and drug targets.