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Chapter 9

Biotechnology and Recombinant DNA

Lectures prepared by Christine L. Case


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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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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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A Typical Genetic Modification Procedure

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Figure 9.1

A Typical Genetic Modification Procedure

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Figure 9.1

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Table 9.2

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Table 9.2

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

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Restriction Enzymes
Cut specific sequences of DNA Destroy bacteriophage DNA in bacterial cells

ANIMATION: Recombinant DNA Technology


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Table 9.1

Restriction Enzyme & Recombinant DNA

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Figure 9.2

Vectors
Carry new DNA to desired cell Plasmids and viruses can be used as vectors

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A Plasmid Vector Used for Cloning

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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
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PCR

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Figure 9.4

PCR

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Figure 9.4

PCR

ANIMATION PCR: Process


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Figure 9.4

Techniques of Genetic Modification

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Inserting Foreign DNA into Cells


DNA can be inserted into a cell by
Electroporation Transformation Protoplast fusion

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Figure 9.5b

Process of Protoplast Fusion

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Figure 9.5a

Inserting Foreign DNA into Cells


DNA can be inserted into a cell by
Gene gun Microinjection

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Transformation

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A Gene Gun

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Figure 9.6

Microinjection of Foreign DNA

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Figure 9.7

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

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Figure 9.8

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

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Figure 9.9

Obtaining DNA
Synthetic DNA is made by a DNA synthesis machine

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Figure 9.10

Selecting a Clone

Figure 9.11
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Selecting a Clone

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Figure 9.11

Selecting a Clone

Figure 9.12
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Selecting a Clone

Figure 9.12
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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

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Figure 9.13

Making a Product
Saccharomyces Mammalian cells cerevisiae May express Used because it is easily eukaryotic genes grown and its genomics easily are known Harder to grow May express eukaryotic genes easily Plant cells and whole plants May express eukaryotic genes easily Plants easily grown
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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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Scientific Applications
Understanding DNA Sequencing organisms' genomes DNA fingerprinting for identification

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Figure 9.17

Southern Blotting

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Figure 9.16

Southern Blotting

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Figure 9.16

Southern Blotting

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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

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