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Genetic Frontiers

Genetic Frontiers. Chapter 15. LEARNING OBJECTIVE 1. Define genetic engineering Outline the primary techniques used in recombinant DNA technology , including genetic probes and DNA cloning. KEY TERMS. GENETIC ENGINEERING Manipulation of genes, often through recombinant DNA technology

robert-pickett
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Genetic Frontiers

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  1. Genetic Frontiers Chapter 15

  2. LEARNING OBJECTIVE 1 • Define genetic engineering • Outline the primary techniques used in recombinant DNA technology, including genetic probes and DNA cloning

  3. KEY TERMS • GENETIC ENGINEERING • Manipulation of genes, often through recombinant DNA technology • RECOMBINANT DNA TECHNOLOGY • Techniques used to make DNA molecules by combining genes from different organisms

  4. Genetic Engineering

  5. Recombinant DNA Technology 1 • DNA molecules are cleaved at specific base sequences • to break them into smaller fragments • Segments of DNA from different sources are joined, forming recombinant DNA molecule • these molecules are taken into a host cell

  6. Recombinant DNA Technology 2 • Cells that take up the gene are identified with a genetic probe • The gene may be transcribed and translated within the host cell • leading to production of a protein not previously produced by the host organism

  7. KEY TERMS • GENETIC PROBE • A single-stranded nucleic acid used to identify a complementary sequence by base pairing with it

  8. Genetic Probe

  9. Bacterial colonies Filter paper 1 Bacterial cells are spread on solid nutrient medium so that only one cell is found in each location. Each cell multiplies to give rise to genetically identical descendants that form a colony on the medium. A few cells from each colony are transferred to special filters. 1 Filter with bacteria from colonies; cells are broken to expose the DNA Radioactively labeled nucleic acid probe is added 2 Radioactively labeled probe nucleic acid is added to the filter; the probe nucleic acid is single stranded and contains a sequence of nucleotides complementary to the gene of interest. 2 3 Some radioactive nucleic acid probe molecules form base pairs with the DNA of some colonies 3 Some of the probe nucleic acid forms base pairs with the DNA of some of the colonies. Exposed X-ray film; dark spots identify colonies with desired DNA 4 DNA from bacterial cells that contain the DNA sequence complementary to the radioactive probe can be detected by X-ray film. 4 Fig. 15-6, p. 299

  10. KEY TERMS • DNA CLONING • Process of selectively amplifying DNA sequences so their structure and function can be studied

  11. LEARNING OBJECTIVE 2 • Explain the actions and importance of restriction enzymes and ligase

  12. KEY TERMS • RESTRICTION ENZYME • An enzyme used in recombinant DNA technology to cleave DNA at specific base sequences • Breaks the DNA molecule into more manageable fragments

  13. Ligase • Segments of DNA from different sources are joined by the enzyme ligase

  14. Restriction Enzymes

  15. Site of cleavage C T T G A A A A G T T C Site of cleavage Sticky end C T A A G T A G T C A T Sticky end Fig. 15-2, p. 295

  16. LEARNING OBJECTIVE 3 • Identify the role of biological vectors, such as plasmids, in recombinant DNA technology • Describe a biological vector and a nonbiological method used to introduce genes into plant cells

  17. KEY TERMS • VECTOR • An agent, such as a plasmid or virus, that transfers DNA from one organism to another • PLASMID • A small, circular DNA molecule that carries genes separate from the main DNA of a bacterial cell

  18. Plasmid

  19. Main bacteria DNA Bacterium Plasmid Fig. 15-3, p. 296

  20. A Biological Vector • The plasmid of Agrobacterium is an effective vector for introducing genes into many plant cells

  21. Constructing Recombinant DNA

  22. DNA from another organism Sticky end Sticky end . . . treated with a restriction enzyme Plasmid from a bacterium . . . Treated with the same restriction enzyme A plasmid and plant DNA are spliced together with DNA ligase Fig. 15-4, p. 297

  23. DNA from another organism Sticky end Sticky end . . . treated with a restriction enzyme Plasmid from a bacterium . . . Treated with the same restriction enzyme A plasmid and plant DNA are spliced together with DNA ligase Stepped Art Fig. 15-4, p. 297

  24. Identifying Bacteria With Altered Plasmids

  25. Sites of cleavage Fragment 2 Fragment 3 Fragment 1 Fragment 4 Plant DNA Cut with a restriction enzyme 1 1 DNA from plant cells is cut into multiple fragments with a restriction enzyme. (Only a small part of one chromosome is shown.) Produce recombinant DNA 2 2 2 2 Recombinant plasmids are formed by cutting plasmids with the same restriction enzyme, mixing the plasmids with the segments of plant DNA, and treating with ligase. 2 Gene for resistance to antibiotic 3 3 Because the recombinant plasmids retain a gene for resistance to an antibiotic (R), bacterial cells that contain the plasmids are resistant to that antibiotic. Plate with antibiotic-containing medium The bacteria are then grown on an antibiotic-containing nutrient medium, and only those that contain the recombinant plasmid survive. 4 4 Bacteria without plasmid fail to grow Bacteria with plasmid live and multiply Fig. 15-5, p. 298

  26. A Nonbiological Method • A nonbiological approach to introduce DNA into plant cells is a genetic “shotgun” • Researchers coat microscopic gold or tungsten fragments with DNA and then shoot them into plant cells

  27. Genetic Engineering

  28. Plasmid Foreign gene Antibiotic-resistant gene Genetically engineered plant cells Cultured plant cells Pieces of plant tissue are placed in a suitable medium, and the cells grow to form a clump of undifferentiated cells. 1 Foreign DNA is spliced into the crown gall plasmid. The recombinant plasmid is inserted into Agrobacterium tumefaciens, which infects plant cells in culture. The plant cells divide in tissue culture. Each cultured plant cell contains the foreign gene. Genetically engineered plants are produced from the cultured plant cells through the use of plant tissue culture techniques. 2 3 4 Fig. 15-8, p. 301

  29. LEARNING OBJECTIVE 4 • Define DNA sequencing

  30. KEY TERMS • DNA SEQUENCING • Procedure by which the sequence of nucleotides in DNA is determined

  31. DNA Sequencing • Automated DNA-sequencing machines connected to powerful computers let scientists sequence huge amounts of DNA quickly and reliably

  32. LEARNING OBJECTIVE 5 • Define genome • Briefly describe the emerging field of genomics

  33. KEY TERMS • GENOME • All the genetic material contained in an individual

  34. KEY TERMS • GENOMICS • Field of biology that studies the genomes of various organisms • Tries to identify all the genes, determine their RNA or protein products, and ascertain how the genes are regulated

  35. LEARNING OBJECTIVE 6 • Explain how RNA interference is used to study gene function

  36. KEY TERMS • RNA INTERFERENCE(RNAi) • Makes use of certain small RNA molecules that interfere with the expression of genes or their RNA transcripts

  37. RNA Interference (RNAi) • After a protein-coding gene is identified, the function of that gene is studied using RNAi to shut the gene off • After the gene is silenced, biologists observe changes in phenotype to determine function of missing protein

  38. LEARNING OBJECTIVE 7 • Describe at least one application of recombinant DNA technology in each of the following: medicine and pharmacology, DNA fingerprinting, and transgenic organisms, specifically genetically modified crops

  39. Medicine and Pharmacology • Escherichia coli have been genetically engineered to produce human insulin • Significant medical benefits to insulin-dependent diabetics

  40. DNA Fingerprinting • Analysis of DNA from an individual • Applications • Investigating crime (forensic analysis) • Studying endangered species in conservation biology • Clarifying disputed parentage

  41. KEY TERMS • TRANSGENIC ORGANISM • A plant or other organism that has foreign DNA incorporated into its genome • GENETICALLY MODIFIED (GM) CROP • A crop plant that has had its genes intentionally manipulated (transgenic crop plant)

  42. Genetically Modified (GM) Crops • Agricultural geneticists developing GM plants that are resistant to insect pests, viral diseases, drought, heat, cold, herbicides, and salty or acidic soil

  43. GM Crops

  44. LEARNING OBJECTIVE 8 • Discuss safety issues associated with recombinant DNA technology • Explain how these issues are being addressed

  45. Safety Issues 1 • Concerns: Genetically engineered organisms might be dangerous if they escaped into the environment • Scientists carried out risky experiments in facilities designed to hold pathogenic organisms

  46. Safety Issues 2 • So far, there is no evidence that researchers have accidentally cloned hazardous genes or released dangerous organisms into the environment • Scientists have relaxed many of restrictive guidelines for using recombinant DNA

  47. Safety Issues 3 • Stringent restrictions still exist where questions about possible effects on the environment are unanswered • (Example: in research that proposes to introduce transgenic organisms into the wild)

  48. Animation: Restriction Enzymes CLICKTO PLAY

  49. Animation: Formation of Recombinant DNA CLICKTO PLAY

  50. Animation: Use of a Radioactive Probe CLICKTO PLAY

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