Biotechnology and Recombinant DNA Chapter 9 Biotechnology and Recombinant DNA

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?

Introduction to Biotechnology Learning Objectives 9-1 Compare and contrast biotechnology, genetic modification, and recombinant DNA technology. 9-2 Identify the roles of a clone and a vector in making recombinant DNA.

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

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

A Typical Genetic Modification Procedure Figure 9.1

A Typical Genetic Modification Procedure Figure 9.1

Table 9.2

Table 9.2

Table 9.3

Differentiate biotechnology and recombinant DNA technology. 9-1 In one sentence, describe how a vector and clone are used. 9-2

Tools of Biotechnology Learning Objectives 9-3 Compare selection and mutation. 9-4 Define restriction enzymes, and outline how they are used to make recombinant DNA. 9-5 List the four properties of vectors. 9-6 Describe the use of plasmid and viral vectors. 9-7 Outline the steps in PCR, and provide an example of its use.

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

Restriction Enzymes Cut specific sequences of DNA Destroy bacteriophage DNA in bacterial cells Cannot digest (host) DNA with methylated cytosines ANIMATION: Recombinant DNA Technology

Table 9.1

Restriction Enzyme & Recombinant DNA Figure 9.2

Vectors Carry new DNA to desired cell Shuttle vectors can exist in several different species Plasmids and viruses can be used as vectors

A Plasmid Vector Used for Cloning Figure 9.3

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: Overview ANIMATION PCR: Components

PCR Figure 9.4

PCR Figure 9.4

PCR ANIMATION PCR: Process Figure 9.4

How are selection and mutation used in biotechnology? 9-3 What is the value of restriction enzymes in recombinant DNA technology? 9-4 What criteria must a vector meet? 9-5 Why is a vector used in recombinant DNA technology? 9-6 For what is each of the following used in PCR: primer, DNA polymerase, 94°C? 9-7

Techniques of Genetic Modification Learning Objectives 9-8 Describe five ways of getting DNA into a cell. 9-9 Describe how a genomic library is made. 9-10 Differentiate cDNA from synthetic DNA. 9-11 Explain how each of the following is used to locate a clone: antibiotic-resistance genes, DNA probes, gene products. 9-12 List one advantage of modifying each of the following: E. coli, Saccharomyces cerevisiae, mammalian cells, plant cells.

Inserting Foreign DNA into Cells DNA can be inserted into a cell by Electroporation Transformation Protoplast fusion Figure 9.5b

Process of Protoplast Fusion Figure 9.5a

Inserting Foreign DNA into Cells DNA can be inserted into a cell by Gene gun Microinjection

A Gene Gun Figure 9.6

Microinjection of Foreign DNA Figure 9.7

Obtaining DNA Genomic libraries are made of pieces of an entire genome stored in plasmids or phages Figure 9.8

Obtaining DNA Complementary DNA (cDNA) is made from mRNA by reverse transcriptase Figure 9.9

Obtaining DNA Synthetic DNA is made by a DNA synthesis machine Figure 9.10

Selecting a Clone Figure 9.11

Selecting a Clone Figure 9.11

Selecting a Clone Figure 9.12

Selecting a Clone Figure 9.12

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

Making a Product Saccharomyces cerevisiae Mammalian cells 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

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?

Contrast the five ways of putting DNA into a cell. 9-8 What is the purpose of a genomic library? 9-9 Why isn’t cDNA synthetic? 9-10 How are recombinant clones identified? 9-11 What types of cells are used for cloning rDNA? 9-12

Applications of rDNA Learning Objectives 9-13 List at least five applications of rDNA technology. 9-14 Define RNAi. 9-15 Discuss the value of the Human Genome Project. 9-16 Define the following terms: random shotgun sequencing, bioinformatics, proteomics.

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

RNA Interference (RNAi) Figure 9.14

Random Shotgun Sequencing Figure 9.15

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

Explain how rDNA technology can be used to treat disease and to prevent disease. 9-13 What is gene silencing? 9-14 How are shotgun sequencing, bioinformatics, and proteomics related to the Human Genome Project? 9-15, 9-16

Applications of rDNA Learning Objectives 9-17 Diagram the Southern blotting procedure, and provide an example of its use. 9-18 Diagram DNA fingerprinting, and provide an example of its use. 9-19 Outline genetic engineering with Agrobacterium.

Scientific Applications Understanding DNA Sequencing organisms' genomes DNA fingerprinting for identification Figure 9.17

Southern Blotting Figure 9.16

Southern Blotting Figure 9.16

Southern Blotting Figure 9.16

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

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

Norovirus Outbreak RT-PCR with a norovirus primer Clinical Focus, p. 266

Nanotechnology Bacteria can make molecule-sized particles Figure 9.18

Using Agrobacterium Bt toxin Herbicide resistance Suppression of genes Antisense DNA Nutrition Human proteins Figure 9.19

Using Agrobacterium Figure 9.20

What is Southern blotting? 9-17 Why do RFLPs result in a DNA fingerprint? 9-18 Of what value is the plant pathogen Agrobacterium? 9-19

Safety Issues and Ethics of Using rDNA Learning Objective 9-20 List the advantages of, and problems associated with, the use of genetic modification techniques.

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?

Identify two advantages and two problems associated with genetically modified organisms. 9-20