Lecture 11 Recombinant Dna Tech


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Lecture 11 Recombinant Dna Tech

  1. 1. Recombinant DNA technology
  2. 2. Essential vocabulary <ul><li>Biotechnology/ GE </li></ul><ul><li>RDNA/ RDNA tech </li></ul><ul><li>Restriction enzymes/endonucleases </li></ul><ul><li>Cloning </li></ul><ul><li>DNA ligase </li></ul><ul><li>Vectors </li></ul><ul><li>Plasmid </li></ul><ul><li>Bacteriophages </li></ul><ul><li>DNA library </li></ul>
  3. 3. Biotechnology <ul><li>The manipulation of biological processes and/or organisms for the benefit of mankind </li></ul>.
  4. 4. RDNA <ul><li>DNA that has been created artificially (not natural). DNA from two or more sources is incorporated into a single recombinant molecule </li></ul>
  5. 5. RDNA tech <ul><li>Procedures by which DNA from different species can be isolated, cut and spliced together </li></ul><ul><li>New &quot;recombinant &quot; molecules are then multiplied in quantity in populations of rapidly dividing cells (e.g. bacteria, yeast). </li></ul><ul><li>Uses methods derived from biochemistry of nucleic acids coupled with genetic techniques originally used for the study of bacteria and viruses </li></ul>
  6. 6. RDNA tech process <ul><li>1. A gene is located on a chromosome map (RFLP) </li></ul><ul><li>2. A DNA library is constructed – plasmid are obtained, cleavage occurs and RDNA prod. </li></ul><ul><li>3. The gene of interest is isolated (cloned) from the library via plasmid DNA isolation, restriction digestion and electrophoresis. </li></ul><ul><li>4. Multiple copies of gene interest are produced for study. </li></ul>
  7. 7. steps <ul><li>Restriction enzymes “cuts” DNA </li></ul><ul><li>at specific sites (restriction fragments) </li></ul><ul><li>DNA ligase “pastes” the DNA </li></ul><ul><li>fragments together </li></ul>The cut segments are inserted into other DNA molecules that serves as vectors
  8. 8. Vectors <ul><li>Carrier DNA mol = transfers the RDNA into the host cell. </li></ul><ul><li>Within host cells, vectors can replicate producing many DNA segments = identical copies (CLONES) </li></ul><ul><li>Host cells pass the recombinant DNA mol on their progeny = population of cells. </li></ul><ul><li>Cloned DNA segments recovered from host cells for purification and analysis </li></ul>
  9. 9. Plasmids <ul><li>Molecules of DNA that are found in bacteria </li></ul><ul><li>Act as a system to transfer genetic material to other bacteria, allowing those to express the transmitted genes. </li></ul><ul><li>small (a few thousand base pairs) </li></ul><ul><li>usually carry only one or a few genes </li></ul><ul><li>circular </li></ul><ul><li>have a single origin of replication </li></ul>
  10. 10. Resctriction enzymes <ul><li>DNA cutting enzymes. Restriction endonucleases </li></ul><ul><li>cuts DNA at a specific site defined by a sequence of bases in the DNA (recog.site) forming “sticky ends” (ss) </li></ul><ul><li>Palindromic sites </li></ul><ul><li>Eg. BamHI cuts 5' GGATCC 3' 3' CCTAGG5’ Eg.2 HaeIII cuts 5'GGCC3' 3'CCGG5' </li></ul><ul><li>several hundred endonucleases have been extracted from bacteria and many are used in recombinant DNA research. eg EcoR1,Hind III, HaeIII, TaqA1, Sau3A </li></ul>
  11. 12. Specific palindromic sites
  12. 15. Library construction <ul><li>Because each cloned DNA segemnt is relatively small, many separate clones must be constructed . </li></ul><ul><li>A set of cloned DNA segments derived from a single individual represents a library </li></ul><ul><li>Cloned libraries could be an entire genome, a singe chromosome, or a set og genes compiled together in a single cell type. </li></ul>
  13. 17. summary
  14. 18. <ul><li>Applications of recombinant DNA technology </li></ul>Used widely in research and hospital laboratories. Broad applications - medicine, agriculture,
  15. 19. ??.. <ul><li>1. The processes of inheritance and gene expression. </li></ul><ul><li>2. Process and tx of various diseases </li></ul><ul><li>3. Generation of economic benefits eg. improved agricultural products. </li></ul>
  16. 20. Applications <ul><ul><li>Molecular Biology/Research </li></ul></ul><ul><ul><li>Diagnostics </li></ul></ul><ul><ul><li>Genetic Counseling </li></ul></ul><ul><ul><li>Criminology/Forensics </li></ul></ul><ul><ul><li>Paternity testing </li></ul></ul><ul><ul><li>Archeology </li></ul></ul><ul><ul><li>Food testing </li></ul></ul><ul><ul><li>Evolutionary studies </li></ul></ul>
  17. 21. Medicine <ul><li>Medicine – production of industrial and commercial compounds </li></ul><ul><li>Insulin – Diabetes </li></ul><ul><li>drugs (angiostation and endostatin) , </li></ul><ul><li>Factor VIII – Haemophilia A </li></ul><ul><li>Factor IX – Haemophilia B </li></ul><ul><li>EPO – Anaemia </li></ul><ul><li>Interleukins and interferons </li></ul><ul><li>Tissue plasminogen activator – dissolve blood clots </li></ul><ul><li>Hormones = GH, parathyroid </li></ul><ul><li>Oxytocin </li></ul><ul><li>Adenosine deaminase –sev. Com. Imm (SCID) </li></ul>,
  18. 22. <ul><li>Diagnostic kits – Hep, AIDS </li></ul><ul><li>Antibiotics </li></ul><ul><li>genetic testing, –Mapping the chromosomal location of genetic disorders. RFLP, DNA fingerprinting – the HG project </li></ul><ul><li>Gene therapy – manipulation of DNA to tx diseases by altering individuals genes . (CF, PKU, DMD) ETHICAL ISSUES </li></ul><ul><li>Forensic applications - All individuals are genetically unique = a distinct &quot;genetic fingerprint“, all types of specs, old and new </li></ul><ul><li>Animals = models of genetic diseases Eg GE mice </li></ul>
  19. 23. Vaccines <ul><li>Vaccines :v are created by transferring the genes that determine a pathogen's surface configuration to a microorganism. </li></ul><ul><li>When the GM microorganism is used in a vaccine, its surface stimulates the production of antibodies. </li></ul><ul><li>These antibodies protects an individual against the pathogen. </li></ul><ul><li>Successful vaccines have been produced for influenza, cold sores, and hepatitis B(HBsAg). </li></ul>
  20. 24. Animals <ul><li>Models of GD : GA to be vulnerable to a disease so that the mechanism of the disease can be investigated and potential therapeutic agents tested. </li></ul><ul><li>Eg. mice GA with a cancer-predisposing gene. Research to test the consequences of exposure to various potential carcinogens and to test the efficacy of preventive drugs. </li></ul>
  21. 25. Agriculture <ul><li>Animals – improved quality/quantity of meat products </li></ul><ul><li>Plants – Gene altered plants : improved crops – quantity, quality and pest resistance. Eg golden rice – GM to contain beta carotene – vit A deficiency. </li></ul>
  22. 26. <ul><li>Spoilage of delicate products. Eg tomatoes if ripened are too soft for shipping. In the past, tomatoes were harvested while green and hard, refrigerated while shipping, and artificially ripened in ethylene gas. </li></ul><ul><li>The softening of tomatoes is caused by the enzyme polygalacturonase. </li></ul><ul><li>Now = The polygalacturonase gene has been inserted, in reverse order, into tomato plants. This inactivates the original enzyme-producing gene and produces a reduction in softening of the tomatoes. </li></ul><ul><li>GA tomatoes can be shipped without expensive refrigeration, which allows them to be produced more cheaply and sold at lower price. </li></ul>
  23. 27. Agriculture <ul><li>Environment. Many waste products of agriculture/industry do not break down naturally/break down slowly. PROBLEM. </li></ul><ul><li>Many bacteria have been GE capable of breaking down oil and other organic wastes </li></ul><ul><li>Cheese making industry : GE Saccharomyces cerevisiae able to dispose of whey by converting lactose to alcohol. </li></ul><ul><li>Agricultural waste products, eg. corn husks, contain cellulose that normally decomposes slowly. Can be converted into sugar by cellulase. Cellulase has been inserted in E.coli making it useful in waste management/disposal programs.. </li></ul>