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  1. Introduction
    1. Genetic engineering-the deliberate modification of an organism's genetic information by directly changing its nucleic acid
    2. Recombinant DNA technology-the collection of methods used to accomplish genetic engineering
  2. Historical Perspectives
    1. Arber and Smith (late 1960s) discovered restriction endonucleases, which cleave DNA at specific sequences; Boyer (1969) first isolated the restriction endonuclease EcoRI
    2. Baltimore and Temin (1970) independently discovered reverse transcriptase; this enzyme can be used to construct a DNA copy, called complementary DNA (cDNA), of any RNA molecule
    3. Jackson, Symons, and Berg (1972) generated the first recombinant DNA molecules by using DNA ligase to join DNA fragments together; Cohen and Boyer (1973) produced the first recombinant plasmid (vector), which was introduced into and replicated within a bacterial host
    4. Southern (1975) developed a blotting procedure for detecting specific DNA fragments, using radioactive DNA hybridization probes; this is useful in isolating particular genes of interest; nonradioactive, enzyme-linked probes can now replace the earlier radioactive probes; they are faster and safer, but may be less sensitive
    5. By the late 1970s, procedures for rapidly sequencing DNA molecules, synthesizing oligonucleotides, and expression of eucaryotic genes in bacteria had been developed
  3. Synthetic DNA
    1. Oligonucleotides-short pieces of DNA or RNA; DNA oligonucleotides can be made by adding one nucleotide at a time to a growing chain
    2. In site-directed mutagenesis, a small synthetic oligonucleotide containing the desired sequence change is used as a primer for DNA polymerase, which then replicates the remainder of the target gene and produces a new gene copy with the desired mutation; this can then be introduced into a new host
  4. The Polymerase Chain Reaction (PCR)
    1. PCR is used to synthesize large quantities of a DNA fragment without cloning it
    2. Synthetic DNA molecules with sequences identical to those flanking the target sequence are used as primers for DNA synthesis; replication is carried out in successive cycles using a heat-stable DNA polymerase
    3. Since its initial discovery, PCR has been automated and improved (e.g., new procedures allow RNA to be used as a template to produce and amplify complementary DNA)
    4. PCR has proven valuable in molecular biology, medicine (e.g., PCR-based diagnostic tests) and in biotechnology (e.g., use of DNA fingerprinting in forensic science)
  5. Preparation of Recombinant DNA
    1. Isolating and cloning fragments
      1. DNA fragments are generated by shearing or by restriction endonuclease cleavage
      2. DNA fragments are separated electrophoretically and the desired fragment is located by the Southern blotting technique
      3. Fragment is inserted into plasmid vector
      4. Plasmid is inserted into bacterium by transformation; in the bacterium the plasmid is replicated (note that the same techniques can be used with synthetic DNA)
      5. Alternatively, a genomic library can be made
        1. The DNA of an organism is fragmented by endonuclease cleavage and all resulting fragments are cloned
        2. The clone containing the desired fragment is identified often by using a nucleic acid hybridization probe
        3. The plasmid is extracted, and the fragment is purified
    2. Gene probes
      1. Can be obtained in several ways
        1. cDNA probes-if the gene is expressed in certain tissue, mRNA is obtained from that tissue and reverse transcriptase is used to produce the cDNA probe
        2. Synthesized probes that are 20 nucleotides long or longer can be made in the laboratory if the amino acid sequence (a partial sequence will suffice) is known
        3. Previously cloned genes can be used as probes if they have sufficient sequence homology to the targeted gene
      2. After being obtained, the probe is labeled to aid detection (e.g., 32P)
    3. Isolating and purifying cloned DNA
      1. An appropriate colony (or phage) is picked from a master plate and then propagated
      2. The plasmid (or phage) DNA is extracted and further purified if needed
      3. The desired fragment is cleaved from the cloning vector with restriction endonuclease and then separated by electrophoresis
  6. Cloning Vectors
    1. Small, well-characterized DNA molecules that contain at least one replication origin, can be replicated within the appropriate host, and code for a phenotype that is easily detected
    2. Plasmids-easy to isolate and purify; can be introduced into bacteria by transformation; often bear antibiotic resistance genes that can be used to select recombinants (e.g., PBR322)
    3. Phage vectors-are more conveniently stored for long periods; contain insertion sites that do not interfere with replication when foreign DNA is inserted; often used to make genomic libraries
      1. Recombinant phage DNA can be packaged into viral capsids and used to infect a host cell
      2. Alternatively, host cells can be directly transformed with recombinant phage DNA; this process is known as transfection
    4. Cosmids-plasmids with lambda phage cos sites; cosmids can be packaged into lambda capsids and then manipulated as a phage; cosmids can also exist in the cell like a plasmid; cosmids can be used to clone very large pieces of DNA
    5. Artificial chromosomes-can be yeast or bacterial; have all of the elements necessary to propagate as a chromosome; they can be used to clone DNA fragments from 100kb to 2000kb in length
  7. Inserting Genes into Eucaryotic Cells
    1. Genes of interest can be inserted directly into animal cells by microinjection; if the genes are stably incorporated into fertilized eggs, the resulting organism is called a transgenic animal
    2. Electroporation is a procedure in which target cells are mixed with DNA and are then exposed briefly to high voltage; this works with mammalian cells and plant cell protoplasts
    3. The gene gun is used to shoot DNA-coated microprojectiles into plant and animal cells
    4. Special plasmids or viruses can also be used to insert desired genes into eucaryotic cells
  8. Expression of Foreign Genes in Bacteria
    1. To express a foreign gene in a bacterium, the gene must:
      1. Have a promoter that is recognized by the host RNA polymerase
      2. Have leader sequences that allow for ribosome binding
      3. Be free of introns
    2. Expression vectors are designed to provide many of the above features; in addition they have useful restriction endonuclease sites and regulatory sequences that can be used to control expression of the foreign gene
    3. The problem of introns can be overcome by cloning cDNA rather than genomic DNA
  9. Applications of Genetic Engineering
    1. Medical applications
      1. Production of medically important proteins, including somatostatin, human growth hormone, human insulin, interferon, interleukin-2, blood-clotting factor VIII, monoclonal antibodies (produced in transgenic plants and mice), and synthetic vaccines
      2. Other uses already developed or being investigated include diagnostic probes for certain infectious diseases and genetic disorders, and somatic cell gene therapy for certain genetic disorders and cancers
      3. In the future, transgenic livestock may be used to produce large amounts of human gene products
    2. Industrial applications
      1. Manufacturing protein products by using recombinant microbes as factories
      2. Improvement of bacterial, fungal, and mammalian cell strains used in industrial bioprocesses
      3. Development of new strains for additional bioprocesses
      4. Bacterial degradation of petroleum products (to clean up oil spills) and other toxic materials
    3. Agricultural applications
      1. Introduction of new desirable traits (e.g., increased growth rate) into farm animals
      2. Transfer of nitrogen fixation capabilities to nonlegume crop plants
      3. Rendering plants resistant to environmental stresses
      4. Currently, many crops grown by farmers in the US are genetically modified (e.g., corn, soybeans, cotton, canola, potato, squash, and tomato)
      5. New applications are being explored, including, protecting crops against frost damage and making plants poisonous to insect pests
  10. Social Impact of Recombinant DNA Technology
    1. Benefits are inherent in applications, but many risks and philosophical questions of the technology must be considered
    2. Safety concerns, including the triggering of widespread infections by the environmental release of recombinant strains of E. coli and the transfer of genes from a weakened strain to a hardy one (with subsequent spread of undesirable genes); guidelines for safe use of recombinant organisms have been developed and are overseen by various government agencies; thus far, no obvious negative effects have been observed and there is concern that the guidelines are being relaxed
    3. Ethical and moral concerns, including attempts to reengineer (improve) the human body, the unethical use of genetic information obtained about an individual, and the unscrupulous use of technology to create biological warfare agents
    4. Ecological concerns including possible ecosystem disruption caused by environmental release of recombinant organisms

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