Chapter 9 Biotechnology
Chapter 9 Biotechnology
Chapter 9 Biotechnology
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<strong>Chapter</strong> 9<br />
<strong>Biotechnology</strong><br />
and<br />
Recombinant DNA<br />
<strong>Biotechnology</strong><br />
and<br />
Recombinant<br />
DNA
Q&A<br />
• Interferons are<br />
species specific, so<br />
that interferons to be<br />
used in humans must<br />
be produced in human<br />
cells. Can you think of<br />
a way to increase the<br />
supply of interferons so<br />
that they can be used<br />
to treat diseases?<br />
Copyright © 2010 Pearson Education, Inc.
Objectives<br />
Compare and contrast biotechnology, recombinant DNA technology, and genetic<br />
engineering.<br />
Identify the roles of a clone and a vector in making recombined DNA.<br />
Compare selection and mutation.<br />
Define restriction enzymes, and outline their use to make recombinant DNA.<br />
List some properties of vectors and describe their use.<br />
Outline the steps in PCR and provide an examples of its use.<br />
Describe various different ways of getting DNA into a cell.<br />
Differentiate cDNA from synthetic DNA.<br />
Explain how each of the following are used to locate a clone: antibiotic-resistance<br />
genes, DNA probes, gene products.<br />
Outline advantages of engineering with either E. coli, Saccharomyces cerevisiae,<br />
mammalian cells, or plant cells.<br />
List some advantages of, and problems associated with, the use of genetic<br />
modification techniques.<br />
Copyright © 2010 Pearson Education, Inc.
Terminology and Definitions<br />
• <strong>Biotechnology</strong>: Manipulation (as through<br />
genetic engineering) of living organisms or their<br />
components to produce useful commercial<br />
products<br />
• Recombinant DNA (rDNA) technology:<br />
Insertion or modification of genes to produce<br />
desired proteins<br />
• Genetic engineering: Techniques used to cut up<br />
and join together genetic material (from different<br />
species) and to introduce the result into an<br />
organism in order to change one or more of its<br />
characteristics.<br />
Copyright © 2010 Pearson Education, Inc.
<strong>Biotechnology</strong> Tools<br />
Selection and Mutation<br />
• Artificial selection: Culture a naturally occurring<br />
microbe that produces desired product<br />
• Mutation: Mutagens cause mutations that might<br />
result in a microbe with a desirable trait. Sitedirected<br />
mutagenesis:<br />
• Restriction Enzymes (RE): Molecular scissors<br />
• Cut specific sequences of DNA<br />
• Destroy bacteriophage DNA in bacterial cells<br />
• Methylases protect own DNA by methylating cytosines<br />
ANIMATION: Recombinant DNA Technology<br />
Copyright © 2010 Pearson Education, Inc.
Site of cleavage<br />
Restriction Enzymes<br />
(= Restriction<br />
Endonucleases)<br />
Fig 8-25<br />
Recognition<br />
sequence is<br />
always a<br />
palindrome
Origin and Naming of Restriction Enzymes
Role of Restriction Enzyme in Making<br />
Recombinant DNA Molecules<br />
Figure of the week: Fig 9.2
Cloning Vectors<br />
• are recombinant DNA molecules.<br />
• introduce foreign DNA into host cells<br />
• are self-replicating in large quantities<br />
Plasmids and<br />
viruses are<br />
commonly used<br />
vectors.<br />
Shuttle vectors can<br />
exist in several<br />
different species.<br />
Copyright © 2010 Pearson Education, Inc.
Polymerase Chain Reaction (PCR)<br />
• Makes multiple copies of a piece of DNA<br />
enzymatically<br />
• Used to<br />
• Clone DNA for recombination<br />
• Amplify DNA to detectable levels<br />
• Sequence DNA<br />
• Diagnose genetic disease<br />
• Detect pathogens<br />
PCR Animation<br />
ANIMATION PCR: Overview<br />
ANIMATION PCR: Components<br />
Copyright © 2010 Pearson Education, Inc.
PCR<br />
Figure of the week: Fig 9.4
Inserting Foreign DNA into<br />
Cells<br />
• DNA can be inserted<br />
into a cell by<br />
• Transformation<br />
• Electroporation<br />
• Protoplast fusion<br />
• Microinjection<br />
Fig 9.5
Obtaining DNA<br />
Genomic libraries:<br />
genes stored in plasmids<br />
or phages<br />
The stored genes can be<br />
• natural copies of genes. –<br />
Exons and introns in Eukaryotes!<br />
• made from mRNA by<br />
reverse transcriptase (cDNA).<br />
• synthetic DNA made by a<br />
DNA synthesis machine.
Obtaining DNA<br />
• Complementary<br />
DNA (cDNA) is<br />
made from mRNA by<br />
reverse transcriptase<br />
Fig 9.9<br />
Copyright © 2010 Pearson Education, Inc.
Blue and White Screening Method for Selecting<br />
a Clone (or Recombinant DNA Molecule)<br />
Direct selection of engineered vector via antibioticresistance<br />
markers (ampR) on plasmid vectors.<br />
Vector also contains-galactosidase gene for bluewhite<br />
screening<br />
Desired gene is inserted into the -galactosidase gene<br />
site gene inactivated<br />
Possible outcomes:<br />
1. Bacterial clones contain recombinant vector resistant<br />
to ampicillin and unable to hydrolyze X-gal (white<br />
colonies).<br />
2. Bacterial clones contain vector without the new gene <br />
blue colonies.<br />
3. Bacteria lack vector will not grow.
Fig 9.11<br />
Possible Method to<br />
detect recombinant<br />
bacteria:<br />
Blue–White<br />
Screening
Making a Gene Product<br />
E. coli: prokaryotic workhorse of biotechnology<br />
(easily grown and its genomics well understood).<br />
Need to eliminate endotoxin from products<br />
Cells must be lysed to get product<br />
Yeast: Saccharomyces cerevisiae is eukaryotic<br />
workhorse of biotechnology. Continuous<br />
secretion of gene product.<br />
Mammalian cells: May express eukaryotic genes<br />
easily. Harder to grow.<br />
Plant cells: Easy to grow. May express eukaryotic<br />
genes easily.<br />
Copyright © 2010 Pearson Education, Inc.
Some <strong>Biotechnology</strong> Applications<br />
Diagnostics: PCR and DNA probes can be used<br />
to quickly identify a pathogen in body tissue or<br />
food. (Forensic microbiology)<br />
Gene therapy to replace defective or missing<br />
genes<br />
Pharmaceutical applications<br />
• Hormone and Antibiotics production<br />
• Vaccines (subunit vaccines, DNA vaccines,<br />
nonpathogenic viruses carrying genes for<br />
pathogen's antigens as vaccines)<br />
Copyright © 2010 Pearson Education, Inc.
Transformation<br />
Cloning genes
Forensic Microbiology<br />
• PCR<br />
• Primer for a specific organism<br />
will allow for detection if that<br />
organism is present<br />
• Real-time PCR: Newly made<br />
DNA tagged with a fluorescent<br />
dye; the levels of fluorescence<br />
can be measured after every<br />
PCR cycle<br />
• Reverse-transcription (RT-<br />
PCR): Reverse transcriptase<br />
makes DNA from viral RNA or<br />
mRNA<br />
RT-PCR with a norovirus primer<br />
Copyright © 2010 Pearson Education, Inc.<br />
Clinical Focus, p. 266
Safety Issues and Ethics of Using rDNA<br />
Strict safety standards avoid accidental release of<br />
genetically modified microorganisms.<br />
Some microbes used in cloning have been altered so<br />
that they cannot survive outside the laboratory.<br />
Microorganisms intended for use in the environment<br />
may be modified to contain suicide genes <br />
organisms do not persist in the environment.<br />
Safety and ethical concerns beyond microbiology: Who<br />
will have access to an individual's genetic<br />
information? Are genetically modified crops safe for<br />
release to environment?<br />
Copyright © 2010 Pearson Education, Inc.
A Typical Genetic Modification Procedure<br />
Foundation Figure<br />
Fig 9.1<br />
Fig 9.1<br />
Copyright © 2010 Pearson Education, Inc.
A Typical Genetic Modification Procedure<br />
Fig 9.1<br />
Copyright © 2010 Pearson Education, Inc.
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