Presentation on theme: "Online Counseling Resource YCMOU ELearning Drive… School of Architecture, Science and Technology Yashwantrao Chavan Maharashtra Open University, Nashik."— Presentation transcript:
Online Counseling Resource YCMOU ELearning Drive… School of Architecture, Science and Technology Yashwantrao Chavan Maharashtra Open University, Nashik – 422222, India
OC-SBT053-CP1-05 Introduction Programmes and Courses SEP – SBT053 – Unit 01
School of Science and Technology, Online Counseling Resource… Introduction A single strand of plant or animal DNA may contain tens of thousands of genes, each programmed to produce a specific protein essential for the growth or survival of the organism. The challenge for geneticists is to isolate individual genes and determine their function.
School of Science and Technology, Online Counseling Resource… Genes A gene is the basic physical and functional unit of heredity. Genes work as subunits of DNA. DNA contains a vast chemical information that carries the complete set of instructions for making all the proteins. Each gene contains a particular set of instructions, usually coding for a particular protein.
School of Science and Technology, Online Counseling Resource… How Genes Make Protein? Different genes are activated in different cells, creating the specific proteins that give a particular cell type its character. For a cell to make protein, The information from a gene is copied, base by base, from DNA into new strands of messenger RNA (mRNA). Then mRNA travels out of the nucleus into the cytoplasm, to cell organelles called ribosomes. There, mRNA directs the assembly of amino acids that fold into completed protein molecule.
School of Science and Technology, Online Counseling Resource… Gene Mutation When a gene undergoes mutation, the protein encoded by that gene will be abnormal. Some mutations are silent; they affect neither the structure of the encoded protein nor its function. Other mutations result in an altered protein. In some instances, the protein is normal enough to function, but not well. This is the case of the flawed hemoglobin, the oxygen-carrying protein in the blood that causes sickle-cell anemia. Thus, it is necessary to isolate the gene & study, so that many genetic problems can be overcome as well as by genetic study the quality of Agricultural, Industrial products can be improve.
School of Science and Technology, Online Counseling Resource… Method of Gene Isolation The method used for the isolation of large scale plasmid DNA is an alkaline lysis procedure followed by equilibrium ultracentrifugation in cesium chloride - ethidium bromide gradients. Briefly, cells containing the desired plasmid are harvested by centrifugation, incubated in a lysozyme buffer, and treated with alkaline detergent. Detergent solubilized proteins and membranes are precipitated with sodium acetate, and the lysate is cleared first by filtration of precipitate through cheesecloth and then by centrifugation. The DNA-containing supernatant is transferred to a new tube, and the plasmid DNA is precipitated by the addition of polyethylene glycol and collected by centrifugation.
School of Science and Technology, Online Counseling Resource… Method of Gene Purification The DNA pellet is resuspended in a buffer containing cesium chloride and ethidium bromide, which is loaded into polyallomer tubes and subjected to ultracentrifugation overnight. The ethidium bromide stained plasmid DNA bands, equilibrated within the cesium chloride density gradient after ultracentrifugation, are visualized under long wave UV light and the lower band is removed with a 5 cc syringe. The intercalating ethidium bromide is separated from the DNA by loading the solution onto an equilibrated ion exchange column. The A260 containing fractions are pooled, diluted, and ethanol precipitated, and the final DNA pellet is resuspended in buffer and assayed by restriction digestion as detected on agarose gel electrophoresis.
School of Science and Technology, Online Counseling Resource… Genetic Mapping To construct a physical map, a chromosome is first broken into smaller pieces of DNA. Then clone the pieces in the laboratory, obtaining millions of identical copies of specific DNA segments. Then line up the clones to reflect the order that existed on the original chromosome. Information about the location and known genetic content of these unique and ordered DNA fragments is stored in a computer, while clones of the ordered pieces themselves are stored in laboratory freezers. When genetic linkage maps indicate that a gene lies in a particular region, we can retrieve clones of interest; then clones are used as the raw material for DNA sequencing actually identifying the order of each and every chemical base in the gene.
School of Science and Technology, Online Counseling Resource… What We Learn…………. Genes are subunits of DNA which are responsible for heredity. Genes contain all the chemical information to synthesize specific protein. Mutated genes carry faulty instructions that produce defective product leads to abnormality. For isolation of gene, DNA is treated with cesium chloride, lysozyme buffer etc. After purification by ultracentrifugation, it can be observed by gel electrophoresis. For genetic mapping, the clones are made which helps in identifying the order of gene.