Copyright © 2010 Pearson Education, Inc. Lectures prepared by Christine L. Case Chapter 9 Biotechnology and Recombinant DNA
Copyright © 2010 Pearson Education, Inc. -Compare and contrast biotechnology, genetic modification, and recombinant DNA technology. -Identify the roles of a clone and a vector in making recombinant DNA. -Compare selection and mutation. -Define restriction enzymes, and describe why they are used to make recombinant DNA. -List the four properties of vectors. -Describe the use of plasmid and viral vectors. -Provide an example of when PCR might be used -Describe five ways of getting DNA into a cell. -Describe how a genomic library is made. -Differentiate cDNA from synthetic DNA. -List one advantage of modifying each of the following: E. coli, Saccharomyces cerevisiae, mammalian cells, plant cells. -List and describe the different types and uses of biotechnology application, including Southern blotting, Diagram DNA fingerprinting for Microbial Forensics, Genetic engineering, and Nanotechonlogy Introduction to Biotechnology – Learning Objectives Upon completion of this chapter, you should be able to:
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
Copyright © 2010 Pearson Education, Inc. Biotechnology and Recombinant DNA Vector: Self-replicating DNA used to carry the desired gene to a new cell Example: a plasmid Clone: Population of cells arising from one cell, each carries the new gene
Copyright © 2010 Pearson Education, Inc. Figure 9.1 A Typical Genetic Modification Procedure
Copyright © 2010 Pearson Education, Inc. Figure 9.1 A Typical Genetic Modification Procedure
Copyright © 2010 Pearson Education, Inc. Table 9.2
Copyright © 2010 Pearson Education, Inc. Table 9.2
Copyright © 2010 Pearson Education, Inc. Table 9.3
Copyright © 2010 Pearson Education, Inc. -Selection of mutants -Restriction Enzymes -Recombinant DNA -Vectors -Plasmids -Viral vectors -Transposons -PCR Tools of Biotechnology
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
Copyright © 2010 Pearson Education, Inc. Restriction Enzymes Cut specific sequences of DNA Destroy bacteriophage DNA in bacterial cells (bacteriophages are species specific) Host DNA cannot be digested by restriction enzymes because of methylated cytosines ANIMATION: Recombinant DNA Technology
Copyright © 2010 Pearson Education, Inc. Table 9.1
Copyright © 2010 Pearson Education, Inc. Figure 9.2 Restriction Enzyme & Recombinant DNA
Copyright © 2010 Pearson Education, Inc. Vectors Carry new DNA to desired cell Shuttle vectors can exist in several different species (bacteria, mammalian cells, yeasts, etc.). Plasmids and viruses can be used as vectors
Copyright © 2010 Pearson Education, Inc. Figure 9.3 A Plasmid Vector Used for Cloning
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
Copyright © 2010 Pearson Education, Inc. Figure 9.4 PCR
Copyright © 2010 Pearson Education, Inc. Figure 9.4 PCR
Copyright © 2010 Pearson Education, Inc. Figure 9.4 PCR ANIMATION PCR: Process
Copyright © 2010 Pearson Education, Inc. -Inserting DNA -Copying DNA -Making a product -Therapeutic Applications -Scientific Applications Techniques of Genetic Modification
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
Copyright © 2010 Pearson Education, Inc. Figure 9.5a Process of Protoplast Fusion
Copyright © 2010 Pearson Education, Inc. Inserting Foreign DNA into Cells DNA can be inserted into a cell by Gene gun Microinjection
Copyright © 2010 Pearson Education, Inc. Figure 9.6 A Gene Gun
Copyright © 2010 Pearson Education, Inc. Figure 9.7 Microinjection of Foreign DNA
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, which are then stored inside of a host
Copyright © 2010 Pearson Education, Inc. Figure 9.9 Obtaining DNA Complementary DNA (cDNA) is made from mRNA by reverse transcriptase
Copyright © 2010 Pearson Education, Inc. Figure 9.10 Obtaining DNA Synthetic DNA is made by a DNA synthesis machine
Copyright © 2010 Pearson Education, Inc. Figure 9.11 Selecting a Clone
Copyright © 2010 Pearson Education, Inc. Figure 9.11 Selecting a Clone
Copyright © 2010 Pearson Education, Inc. Figure 9.12 Selecting a Clone
Copyright © 2010 Pearson Education, Inc. Figure 9.12 Selecting a Clone
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
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
Copyright © 2010 Pearson Education, Inc. Therapeutic Applications of rDNA Producing human enzymes and other proteins Creating Subunit vaccines (only contain a protein portion) Nonpathogenic viruses carrying genes for pathogen's antigens as DNA vaccines Gene therapy to replace defective or missing genes Gene silencing Identifying entire genomes Using shotgun sequencing The human genome project
Copyright © 2010 Pearson Education, Inc. Figure 9.14 RNA Interference (RNAi) – a type of gene silencing
Copyright © 2010 Pearson Education, Inc. Figure 9.15 Random Shotgun Sequencing
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
Copyright © 2010 Pearson Education, Inc. Figure 9.17 Scientific Applications Understanding DNA Sequencing organisms' genomes DNA fingerprinting for identification
Copyright © 2010 Pearson Education, Inc. Figure 9.16 Southern Blotting
Copyright © 2010 Pearson Education, Inc. Figure 9.16 Southern Blotting
Copyright © 2010 Pearson Education, Inc. Figure 9.16 Southern Blotting
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
Copyright © 2010 Pearson Education, Inc. Example: A Norovirus Outbreak Are the outbreaks related? What is the source? Clinical Focus, p. 266
Copyright © 2010 Pearson Education, Inc. Nanotechnology Bacteria can make molecule-sized particles or they can provide the much needed metals to construct the tiny machinery necessary in nanotechnology Figure 9.18
Copyright © 2010 Pearson Education, Inc. Using Agrobacterium Bt toxin Herbicide resistance Suppression of genes Antisense DNA Nutrition Human proteins Figure 9.19
Copyright © 2010 Pearson Education, Inc. Using Agrobacterium Figure 9.20
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?