Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Chapter 12

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12.1 A scientific Revolution Genetic engineering is the process of moving genes from one organism to another Having a major impact on agriculture & medicine Increasing yieldsCuring diseaseProducing insulin

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12.2 Restriction Enzymes Restriction enzymes bind to specific short sequences (usually 4- to 6- bases long) on the DNA GAATTC CTTAAG Most restriction enzymes cut the DNA in a staggered fashion This generates “sticky” ends These ends can pair with any other DNA fragment generated by the same enzyme The pairing is aided by DNA ligase The nucleotide sequence on both DNA strands is identical when read in opposite directions

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12.3 The Four Stages of a Genetic Engineering Experiment All gene transfer experiments share four distinct stages 1. Cleaving DNA 2. Producing recombinant DNA 3. Cloning 4. Screening

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1. Cleaving the DNA The large number of fragments produced are separated by electrophoresis Fragments appear as bands under fluorescent light

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 2. Producing Recombinant DNA Fragments of source DNA are inserted into vectors Vectors are plasmids or viruses that carry foreign DNA into the host cell Vector DNA is cut with the same enzyme as the source DNA, thus allowing the joining of the two 3. Cloning Host cells are usually bacteria As each bacterial cell reproduces, it forms a clone of cells containing the fragment-bearing vector Together all clones constitute a clone library

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 4. Screening A preliminary screen of the clone library eliminates 1. Clones without vectors 2. Clones with vectors that do not contain DNA The vector employed usually has genes for a. Antibiotic resistance This eliminates the first type of clones because they are sensitive to antibiotics b.  -galactosidase This eliminates the second type of clones based on X-gal metabolism and color changes

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4. Screening To find the gene of interest, the clone library is screened by a process termed hybridization The cloned genes form base pairs with complementary sequences on another nucleic acid, termed the probe The bacterial colonies are first grown on agar They are then transferred to a filter The filter is treated with a radioactive probe The filter is then subjected to autoradiography

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12.4 Working with DNA Key techniques used by today’s genetic engineers include PCR amplification Used to increase the amounts of DNA cDNA formation Used to build genes from their mRNA DNA fingerprinting Used to identify particular individuals

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The polymerase chain reaction (PCR) requires primers Short single-stranded sequences complementary to regions on either side of the DNA of interest PCR consists of three basic steps 1. Denaturation 2. Primer annealing 3. Primer extension PCR Amplification

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Target sequence Primers Denaturation 1 Heat 2 Annealing of primers Cool 2 copies Free nucleotides 3 Primer extension DNA polymerase Cycle 1 Heat Cool 4 copies Cycle 2 Cool Heat 8 copies Cycle 3

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The primary mRNA transcript contains exons and introns The processed mRNA contains only exons It is used as a template to create a single strand of DNA termed complementary DNA (cDNA) cDNA is then converted to a double-stranded molecule cDNA Formation

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This is a process that is used to determine if two DNA samples are from the same source The DNA from the two sources is fragmented using restriction enzymes The fragments are separated using gel electrophoresis They are transferred to a filter The filters are screened with radioactive probes Then subjected to autoradiography DNA Fingerprinting

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Two of the DNA profiles that lead to conviction First time DNA profiles were used in court of law

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12.5 Genetic Engineering and Medicine Genetic engineering has been used in many medical applications 1. Production of proteins to treat illnesses 2. Creation of vaccines to combat infections 3. Replacement of defective genes Gene therapy is discussed in Chapter 12

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display In diabetes, the body is unable to control levels of sugar in the blood because of lack of insulin Diabetes can be cured if the body is supplied with insulin Making “Magic Bullets” The gene encoding insulin has been introduced into bacteria

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Other genetically engineered drugs include Making “Magic Bullets” Anticoagulants Used to treat heart attack patients Factor VIII Used to treat hemophilia Human growth hormone (HGH) Used to treat dwarfism Has only one extra gene: HGH

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Genetic engineering has also been used to create subunit vaccines against viruses Piggyback Vaccines A gene encoding a viral protein is put into the DNA of a harmless virus and injected into the body The viral protein will elicit antibody production in the animal A novel kind of vaccine was introduced in 1995 The DNA vaccine uses plasmid vectors It elicits a cellular immune response, rather than antibody production

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Constructing a piggyback vaccine for the herpes simplex virus

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12.6 Genetic Engineering of Farm Animals In 1994, the recombinant hormone bovine somatotropin (BST) became commercially available Dairy farmers used BST as a supplement to enhance milk production in cows Consumers are concerned about the presence of the hormone in milk served to children This fear is unfounded

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The production of BST through genetic engineering

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12.7 Genetic Engineering of Crop Plants Successful manipulation of the genes of crop plants has improved the quality of these plants Pest resistance Leads to a reduction in the use of pesticides Bt, a protein produced by soil bacteria, is harmful to pests but not to humans The Bt gene has been introduced into tomato plants, among others

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12.7 Genetic Engineering of Crop Plants Herbicide resistance Crop plants have been created that are resistant to glyphosate Herbicide resistance offers two main advantages 1. Lowers the cost of producing crops 2. Reduces plowing and conserves the top soil Petunias Glyphosate- sensitive plants Glyphosate- resistant plants

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12.7 Genetic Engineering of Crop Plants More Nutritious Crops Worldwide, two major deficiencies are iron and vitamin A Deficiencies are especially severe in developing countries where the major staple food is rice Ingo Potrykus, a Swiss bioengineer, developed transgenic “golden” rice to solve this problem

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Transgenic “golden” rice

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The promise of genetic engineering is very much in evidence However, it has generated considerable controversy and protest Are genetic engineers “playing God” by tampering with the genetic material? Two sets of risks need to be considered 1. Are GM foods safe to eat? 2. Are GM foods safe for the environment? Potential Risks of Genetically Modified (GM) Crops

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Potential Risks of Genetically Modified (GM) Crops 1. Are GM foods safe to eat? The herbicide glyphosate blocks the synthesis of aromatic amino acids Humans don’t make any aromatic amino acids, so glyphosate doesn’t hurt us However, gene modifications that render plants resistant to glyphosate may introduce novel proteins Moreover, introduced proteins may cause allergies in humans

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Potential Risks of Genetically Modified (GM) Crops 2. Are GM foods safe for the environment? Three legitimate concerns are raised 1. Harm to other organisms Will other organisms be harmed unintentionally? 2. Resistance Will pests become resistant to pesticides? 3. Gene flow What if introduced genes will pass from GM crops to their wild or weedy relatives?

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Potential Risks of Genetically Modified (GM) Crops Should GM foods be labeled? Every serious scientific investigation has concluded that GM foods are safe So there is no health need for a GM label However, people have a right to know what is in their food So there may be a need for label after all