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Genetic Engineering: Molecular Biology.


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Genetic Engineering: Molecular Biology.

  1. 1. Foundations in Microbiology Chapter 10 PowerPoint to accompany Fifth Edition Talaro Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
  2. 2. Genetic Engineering: A Revolution in Molecular Biology Chapter 10
  3. 3. Genetic engineering <ul><li>direct, deliberate modification of an organism’s genome </li></ul><ul><li>bioengineering </li></ul><ul><li>Biotechnology – use of an organism’s biochemical and metabolic pathways for industrial production </li></ul>
  4. 4. I. Tools & Techniques of genetic engineering <ul><li>enzymes for dicing, splicing, & reversing nucleic acids </li></ul><ul><li>analysis of DNA </li></ul>
  5. 5. Enzymes for dicing, splicing, & reversing nucleic acids <ul><li>restriction endonucleases – recognize specific sequences of DNA & break phosphodiester bonds </li></ul><ul><li>ligase – rejoins phosphate-sugar bonds cut by endonucleases </li></ul><ul><li>reverse transcriptase – makes a DNA copy of RNA - cDNA </li></ul>
  6. 7. Analysis of DNA <ul><li>gel electrophoresis- separates DNA fragments based on size </li></ul><ul><li>nucleic acid hybridization & probes – probes base pair with complementary sequences; used to detect specific sequences </li></ul><ul><li>DNA Sequencing – reading the sequence of nucleotides in a stretch of DNA </li></ul><ul><li>Polymerase Chain Reaction – way to amplify DNA </li></ul>
  7. 8. Gel electrophoresis
  8. 9. Southern blot hydridization
  9. 10. In situ hybridization
  10. 11. Sanger DNA sequence technique
  11. 12. Polymerase chain reaction (PCR)
  12. 13. II. Methods in Recombinant DNA Technology <ul><li>concerned with transferring DNA from one organism to another </li></ul><ul><li>Cloning vectors & hosts </li></ul><ul><li>Construction of a recombinant plasmid </li></ul>
  13. 15. Characteristics of cloning vectors <ul><li>must be capable of carrying a significant piece of donor DNA </li></ul><ul><li>must be readily accepted by the cloning host </li></ul><ul><li>plasmids – small, well characterized, easy to manipulate & can be transferred into appropriate host cells through transformation </li></ul><ul><li>bacteriophages – have the natural ability to inject their DNA into bacterial hosts through transduction </li></ul>
  14. 16. Vector considerations <ul><li>origin of replication </li></ul><ul><li>size of donated DNA vector will accept </li></ul><ul><li>gene which confers drug resistance to their cloning host </li></ul>
  15. 17. pBR322
  16. 18. Characteristics of cloning hosts <ul><li>rapid overturn, fast growth rate </li></ul><ul><li>can be grown in large quantities using ordinary culture methods </li></ul><ul><li>nonpathogenic </li></ul><ul><li>genome that is well delineated </li></ul><ul><li>capable of accepting plasmid or bacteriophage vectors </li></ul><ul><li>maintains foreign genes through multiple generations </li></ul><ul><li>will secrete a high yield of proteins from expressed foreign genes </li></ul>
  17. 21. III. Biochemical Products of Recombinant DNA Technology <ul><li>enables large scale manufacturing of life-saving hormones, enzymes, vaccines </li></ul><ul><ul><li>insulin for diabetes </li></ul></ul><ul><ul><li>human growth hormone for dwarfism </li></ul></ul><ul><ul><li>erythropoietin for anemia </li></ul></ul><ul><ul><li>Factor VIII for hemophilia </li></ul></ul><ul><ul><li>HBV vaccine </li></ul></ul>
  18. 22. IV. Genetically Modified Organisms (GMO) <ul><li>Recombinant microbes </li></ul><ul><ul><li>Pseudomonas syringae – prevents ice crystals </li></ul></ul><ul><ul><li>Bacillus thuringienisis –encodes an insecticide </li></ul></ul><ul><li>Transgenic plants </li></ul><ul><ul><li>Rice that makes beta-carotene </li></ul></ul><ul><ul><li>Tobacco resistant to herbicides </li></ul></ul><ul><ul><li>Peas resistant to weevils </li></ul></ul><ul><li>Transgenic animals </li></ul><ul><ul><li>Mouse models for CF, Alzheimer’s, sickle cell anemia </li></ul></ul><ul><ul><li>Sheep or goats that make medicine in their milk semen </li></ul></ul>
  19. 23. Bioengineering of plants
  20. 24. Transgenic mice
  21. 25. V. Genetic Treatments <ul><li>Gene therapy </li></ul><ul><li>Antisense DNA </li></ul><ul><li>Triplex DNA </li></ul>
  22. 26. Gene therapy <ul><li>correct faulty gene in human suffering from disease </li></ul><ul><ul><li>ex vivo – normal gene is is added to tissues taken from the body, then transfected cells are reintroduced into the body </li></ul></ul><ul><ul><li>in vivo – naked DNA or viral vector is directly introduced into patient’s tissue </li></ul></ul><ul><li>Most trials target cancer, single gene defects & infections </li></ul><ul><li>Most gene deliveries are carried out by viral vectors </li></ul>
  23. 27. Gene therapy
  24. 28. Antisense DNA: targeting mRNA <ul><li>Antisense – a nucleic acid strand with a base sequence that is complementary to the translatable strand </li></ul><ul><li>Antisense DNA gets into the nucleus and binds to mRNA, blocking the expression of an unwanted protein </li></ul><ul><ul><li>cancers </li></ul></ul><ul><ul><li>Alzheimer’s disease </li></ul></ul><ul><ul><li>autoimmune diseases </li></ul></ul>
  25. 29. Triplex DNA <ul><li>A triple helix formed when a third strand of DNA inserts into the major groove, making it inaccessible to normal transcription </li></ul><ul><li>oligonucleotides have been synthesized to form triplex DNA </li></ul><ul><ul><li>oncogenes </li></ul></ul><ul><ul><li>viruses </li></ul></ul><ul><ul><li>receptor for IL-2 </li></ul></ul>
  26. 30. Antisense DNA & triplex DNA
  27. 31. VI. Genome Analysis <ul><li>Gene Mapping </li></ul><ul><li>DNA Fingerprinting </li></ul><ul><li>Microarray analysis </li></ul>
  28. 32. Gene Mapping <ul><li>determining the location of specific genes on the chromosomes </li></ul><ul><li>Human Genome Project – to determine the nucleotide sequence of the >30,000 genes in the genome & the importance of these sequences & how they relate to human disease </li></ul>
  29. 33. Map of chromosome 16
  30. 34. DNA Fingerprinting <ul><li>Every individual has a unique sequence of DNA </li></ul><ul><li>Used to: </li></ul><ul><ul><li>identify hereditary relationships </li></ul></ul><ul><ul><li>study inheritance of patterns of diseases </li></ul></ul><ul><ul><li>study human evolution </li></ul></ul><ul><ul><li>identify criminals or victims of disaster </li></ul></ul>
  31. 35. DNA fingerprints
  32. 36. Pedigree analysis
  33. 37. Microarray analysis <ul><li>Method of determining which genes are actively transcribed in a cell under various conditions </li></ul><ul><ul><li>health vs disease </li></ul></ul><ul><ul><li>growth vs differentiation </li></ul></ul><ul><li>could improve accuracy of diagnosis and specificity of treatment </li></ul>
  34. 38. Microarray