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Copyright © 2010 Pearson Education, Inc. Lectures prepared by Christine L. Case Chapter 9 Biotechnology and Recombinant DNA
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Copyright © 2010 Pearson Education, Inc. Biotechnology and Recombinant DNA Biotechnology: The use of microorganisms, cells, or cell components to make a product. Foods, antibiotics, vitamins, enzymes Recombinant DNA (rDNA) technology: Insertion or modification of genes to produce desired proteins
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Copyright © 2010 Pearson Education, Inc. Biotechnology and Recombinant DNA Vector: Self-replicating DNA used to carry the desired gene to a new cell Clone: Population of cells arising from one cell, each carries the new gene
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Copyright © 2010 Pearson Education, Inc. Figure 9.1 A Typical Genetic Modification Procedure
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Copyright © 2010 Pearson Education, Inc. Figure 9.1 A Typical Genetic Modification Procedure
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Copyright © 2010 Pearson Education, Inc. Table 9.2
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Copyright © 2010 Pearson Education, Inc. Table 9.2
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Copyright © 2010 Pearson Education, Inc. Table 9.3
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Copyright © 2010 Pearson Education, Inc. Selection and Mutation Selection: Culture a naturally occurring microbe that produces desired product Mutation: Mutagens cause mutations that might result in a microbe with a desirable trait Site-directed mutagenesis: Change a specific DNA code to change a protein Select and culture microbe with the desired mutation
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Copyright © 2010 Pearson Education, Inc. Restriction Enzymes Cut specific sequences of DNA Destroy bacteriophage DNA in bacterial cells Cannot digest (host) DNA with methylated cytosines ANIMATION: Recombinant DNA Technology
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Copyright © 2010 Pearson Education, Inc. Table 9.1
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Copyright © 2010 Pearson Education, Inc. Figure 9.2 Restriction Enzyme & Recombinant DNA
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Copyright © 2010 Pearson Education, Inc. Vectors Carry new DNA to desired cell Shuttle vectors can exist in several different species Plasmids and viruses can be used as vectors
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Copyright © 2010 Pearson Education, Inc. Figure 9.3 A Plasmid Vector Used for Cloning
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Copyright © 2010 Pearson Education, Inc. Polymerase Chain Reaction (PCR) To make multiple copies of a piece of DNA enzymatically Used to Clone DNA for recombination Amplify DNA to detectable levels Sequence DNA Diagnose genetic disease Detect pathogens ANIMATION PCR: Components ANIMATION PCR: Overview
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Copyright © 2010 Pearson Education, Inc. Figure 9.4 PCR
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Copyright © 2010 Pearson Education, Inc. Figure 9.4 PCR
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Copyright © 2010 Pearson Education, Inc. Figure 9.4 PCR ANIMATION PCR: Process
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Copyright © 2010 Pearson Education, Inc. Figure 9.5b Inserting Foreign DNA into Cells DNA can be inserted into a cell by Electroporation Transformation Protoplast fusion
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Copyright © 2010 Pearson Education, Inc. Figure 9.5a Process of Protoplast Fusion
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Copyright © 2010 Pearson Education, Inc. Inserting Foreign DNA into Cells DNA can be inserted into a cell by Gene gun Microinjection
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Copyright © 2010 Pearson Education, Inc. Figure 9.6 A Gene Gun
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Copyright © 2010 Pearson Education, Inc. Figure 9.7 Microinjection of Foreign DNA
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Copyright © 2010 Pearson Education, Inc. Figure 9.8 Obtaining DNA Genomic libraries are made of pieces of an entire genome stored in plasmids or phages
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Copyright © 2010 Pearson Education, Inc. Figure 9.9 Obtaining DNA Complementary DNA (cDNA) is made from mRNA by reverse transcriptase
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Copyright © 2010 Pearson Education, Inc. Figure 9.10 Obtaining DNA Synthetic DNA is made by a DNA synthesis machine
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Copyright © 2010 Pearson Education, Inc. Figure 9.11 Selecting a Clone
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Copyright © 2010 Pearson Education, Inc. Figure 9.11 Selecting a Clone
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Copyright © 2010 Pearson Education, Inc. Figure 9.12 Selecting a Clone
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Copyright © 2010 Pearson Education, Inc. Figure 9.12 Selecting a Clone
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Copyright © 2010 Pearson Education, Inc. Making a Product E. coli Used because it is easily grown and its genomics are known Need to eliminate endotoxin from products Cells must be lysed to get product Figure 9.13
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Copyright © 2010 Pearson Education, Inc. Making a Product Saccharomyces cerevisiae Used because it is easily grown and its genomics are known May express eukaryotic genes easily Mammalian cells May express eukaryotic genes easily Harder to grow Plant cells and whole plants May express eukaryotic genes easily Plants easily grown
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Copyright © 2010 Pearson Education, Inc. Q&A Interferons are species specific, so that interferons to be used in humans must be produced in human cells. Can you think of a way to increase the supply of interferons so that they can be used to treat diseases?
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Copyright © 2010 Pearson Education, Inc. Therapeutic Applications Human enzymes and other proteins Subunit vaccines Nonpathogenic viruses carrying genes for pathogen's antigens as DNA vaccines Gene therapy to replace defective or missing genes
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Copyright © 2010 Pearson Education, Inc. Figure 9.14 RNA Interference (RNAi)
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Copyright © 2010 Pearson Education, Inc. Figure 9.15 Random Shotgun Sequencing
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Copyright © 2010 Pearson Education, Inc. The Human Genome Project Nucleotides have been sequenced Human Proteome Project may provide diagnostics and treatments Reverse genetics: Block a gene to determine its function
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Copyright © 2010 Pearson Education, Inc. Figure 9.17 Scientific Applications Understanding DNA Sequencing organisms' genomes DNA fingerprinting for identification
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Copyright © 2010 Pearson Education, Inc. Figure 9.16 Southern Blotting
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Copyright © 2010 Pearson Education, Inc. Figure 9.16 Southern Blotting
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Copyright © 2010 Pearson Education, Inc. Figure 9.16 Southern Blotting
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Copyright © 2010 Pearson Education, Inc. Forensic Microbiology PCR Primer for a specific organism will cause application if that organism is present Real-time PCR: Newly made DNA tagged with a fluorescent dye; the levels of fluorescence can be measured after every PCR cycle Reverse-transcription (RT-PCR): Reverse transcriptase makes DNA from viral RNA or mRNA
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Copyright © 2010 Pearson Education, Inc. Norovirus Outbreak Are the outbreaks related? What is the source? Clinical Focus, p. 266
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Copyright © 2010 Pearson Education, Inc. Norovirus Outbreak RT-PCR with a norovirus primer Clinical Focus, p. 266
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Copyright © 2010 Pearson Education, Inc. Nanotechnology Bacteria can make molecule-sized particles Figure 9.18
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Copyright © 2010 Pearson Education, Inc. Using Agrobacterium Bt toxin Herbicide resistance Suppression of genes Antisense DNA Nutrition Human proteins Figure 9.19
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Copyright © 2010 Pearson Education, Inc. Using Agrobacterium Figure 9.20
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Copyright © 2010 Pearson Education, Inc. Safety Issues and Ethics of Using rDNA Avoid accidental release Genetically modified crops must be safe for consumption and for the environment Who will have access to an individual's genetic information?
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