Copyright © 2010 Pearson Education, Inc. Lectures prepared by Christine L. Case Chapter 9 Biotechnology and Recombinant DNA

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  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 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  Cannot digest (host) DNA with 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  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. 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

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. 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?

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

Copyright © 2010 Pearson Education, Inc. Figure 9.14 RNA Interference (RNAi)

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. Norovirus Outbreak  Are the outbreaks related?  What is the source? Clinical Focus, p. 266

Copyright © 2010 Pearson Education, Inc. Norovirus Outbreak  RT-PCR with a norovirus primer Clinical Focus, p. 266

Copyright © 2010 Pearson Education, Inc. Nanotechnology  Bacteria can make molecule-sized particles 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?