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