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Microbiology, 4/e Prescott, Harley, Klein | ||||||
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15 Recombinant DNA Technology
CHAPTER OVERVIEW
This chapter focuses on practical applications of microbial genetic principles discussed in previous chapters. Although we have been altering the genetic makeup of organisms for centuries and nature has been doing it even longer, only recently have we been able to manipulate the DNA directly using genetic engineering or recombinant DNA technology. The potential benefits of these techniques are great and affect such diverse areas as medicine, agriculture, and industry. However, the use of these techniques is not without risks, and these risks must be considered in any discussion of this technology.
CHAPTER OBJECTIVES
After reading this chapter you should be able to:
o discuss the use of recombinant DNA technology to genetically engineer various organisms
o discuss the key role played by restriction endonucleases in genetic engineering
o discuss how plasmids, phages, and cosmids are used as vectors for insertion and expression of foreign genes in an organism
o discuss the use of both procaryotes and eucaryotes as target organisms for foreign gene insertion
o discuss the contributions already made by the use of this technology
o discuss the risks and the ethical problems associated with the use of this technology
CHAPTER OUTLINE
I. Historical Perspectives
A. Arber and Smith (late 1960s) discovered restriction endonucleases, which cleave DNA at specific sequences
B. Boyer (1969) first isolated the restriction endonuclease EcoRI
C. 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
D. Jackson, Symons, and Berg (1972) generated the first recombinant DNA molecules
E. Cohen and Boyer (1973) produced the first recombinant plasmid (vector), which was introduced into and replicated within a bacterial host
F. Southern (1975) developed the blotting procedure for detecting specific DNA fragments, which uses radioactive DNA hybridization probes; this is useful in isolating particular genes of interest
G. Maxam and Gilbert (and independently, Sanger) (1976-1978) developed procedures for rapidly sequencing DNA molecules
H. Nonradioactive, enzyme-linked probes can now replace the earlier radioactive probes; they are faster and safer, but may be less sensitive
II. Synthetic DNA
A. Can be made by adding one nucleotide at a time to a growing chain; this takes about 40 minutes per nucleotide
B. 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
III. The polymerase chain reaction (PCR)
A. Synthetic DNAs with sequences identical to those flanking the target sequence (100 to 5,000 base pairs in length) are used as primers; this is followed by successive cycles with a heat-stable DNA polymerase; using this technique, billions of copies of the target sequence can be produced in one to two hours
B. New procedures allow RNA to be used as a template to produce and amplify complementary DNA (cDNA)
C. PCR is now being used in forensic science as part of the DNA fingerprinting process
IV. Preparation of Recombinant DNA
A. Isolating and cloning fragments
1. DNA fragments are generated by shearing or by restriction endonuclease cleavage
2. DNA fragments are separated electrophoretically
3. Desired fragment is located by the Southern blotting technique; there must be a way of specifically identifying the desired fragment
4. Fragment is inserted into plasmid vector
5. Plasmid is inserted into bacterium by transformation
6. Alternatively, fragments can be inserted into phage particles and introduced into bacterium by infection (transduction)
7. Bacteria are grown, replica plated onto nitrocellulose filters, lysed, reacted with a hybridization probe specific for the desired gene, and then the desired clone is identified
B. Alternative strategies
1. Production of a genomic library
a. The DNA is fragmented by endonuclease cleavage
b. All resulting fragments are cloned
c. The clone containing the desired fragment is identified and amplified
d. The plasmid is extracted, and the fragment is purified
2. A synthetic DNA can be produced and then cloned
C. Gene probes
1. cDNA probes can be used if the gene is expressed in certain tissue; the mRNA is obtained 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
D. Isolating and purifying cloned DNA
1. An appropriate colony is picked from a master plate and then propagated
2. The plasmid (or phage) DNA is extracted and purified by gradient centrifugation
3. The desired fragment is cleaved from the DNA with restriction endonuclease and then separated by electrophoresis
V. Cloning Vectors-small, well-characterized DNA molecules that contain at least one replication origin and that can be replicated within the appropriate host
A. Plasmids
1. Easy to isolate and purify
2. Can be introduced into bacteria by transformation
3. Often bear antibiotic resistance genes that can be used to select recombinants
B. Phage vectors are more conveniently stored for long periods, and they contain insertion sites that do not interfere with replication when foreign DNA is inserted
C. Recombinant phage DNA (rather than complete phage particles) can also be used directly, a process known as transfection; however, this approach is less efficient
D. Cosmids are plasmids with lambda phage cos sites and can be packaged into lambda capsids; they can be transmitted similarly to phages but can exist in the cell like a plasmid; these can be used for larger pieces of DNA
E. Shuttle vectors are used to transfer genes between very different organisms; they usually contain one replication origin for each host
F. Artificial chromosomes (yeast or bacterial) have all of the elements necessary to propagate as a chromosome that can be used to clone DNA fragments from 100kb to 2000kb in length
VI. Inserting Genes into Eucaryotic Cells
A. 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
B. 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
C. The gene gun is used to shoot DNA-coated microprojectiles into plant and animal cells
VII. Expression of Foreign Genes in Bacteria
A. A promoter is necessary
B. An appropriate leader is necessary; leaders are different in procaryotes and eucaryotes
C. Introns in eucaryotic genes must be removed because bacteria cannot remove them
D. Regulatory sequences are often used to control expression
E. Other modifications have been used to increase the efficiency of expression, and to facilitate recovery of the protein product
F. Expression vectors are cloning vectors that have been modified for easy insertion and expression of foreign DNA
VIII. Applications of Genetic Engineering
A. Medical applications
1. Somatostatin, a growth regulatory hormone
2. Human growth hormone for treating pituitary dwarfism
3. Human insulin, for people allergic to bovine or porcine insulin
4. Interferon, for treatment of viral infections and possibly cancer
5. Other important polypeptides, including interleukin-2 and blood-clotting factor VIII
6. Transgenic corn and soybeans can be used to produce monoclonal antibodies
7. Genetically engineered mice can now produce fully human monoclonal antibodies
8. Synthetic vaccines (investigational)
9. Diagnostic probes for certain genetic disorders (investigational)
10. Somatic cell gene therapy for certain genetic disorders (investigational, but has been recently attempted in a patient with an immune deficiency disease with the use of a modified retrovirus)
11. The use of transgenic livestock to produce large amounts of human gene products (investigational)
12. Fusion toxins have been produced that target certain cells to deliver the toxin
B. Industrial applications
1. Improvement of bacterial, fungal, and mammalian cell strains used in industrial bioprocesses
2. Development of new strains for additional bioprocesses
3. Bacterial metabolism of petroleum products (to clean up oil spills)
4. Bacterial metabolism of other toxic materials
C. 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. Rendering fruits and vegetables less susceptible to rotting so that they can be left to ripen longer; the first such food, the Flavr Savr tomato, has been approved by the FDA for sale
5. Protecting crops against frost damage
6. Making plants poisonous to insect pests
7. Increasing milk production in dairy cattle
IX. Social Impact of Recombinant DNA Technology
A. Benefits are inherent in applications
B. It is possible that environmental release might trigger widespread infections
C. The transfer of genes from a weakened strain to a hardy one (with subsequent spread of undesirable genes) is a potential problem
D. Unethical use of information obtained about an individual; this is a concern within the human genome project which will improve the process of genetic screening for disorders
E. The unscrupulous use of technology to create biological warfare agents must be considered
F. Eugenic application in germ line cells could cause problems
G. Ecosystem disruption could result from environmental release
H. Guidelines for safe use have been developed and are overseen by various government agencies
I. Thus far, no obvious negative ecological effects have been observed, and currently, safety guidelines are being somewhat relaxed