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Chapter 9
Biotechnology
and
Recombinant DNA
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?
Copyright © 2010 Pearson Education, Inc.
Objectives
Compare 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.
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Selection
and Mutation
Biotechnology
Tools
 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. Sitedirected mutagenesis:
 Restriction Enzymes (RE): Molecular scissors
 Cut specific sequences of DNA
 Destroy bacteriophage DNA in bacterial cells
 Methylases protect own DNA by methylating cytosines
ANIMATION: Recombinant DNA Technology
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Site of cleavage
Restriction Enzymes
(= Restriction
Endonucleases)
Fig 8-25
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
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
PCR Animation
 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
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.
Obtaining DNA
 Complementary
DNA (cDNA) is
made from mRNA by
reverse transcriptase
Fig 9.9
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Blue and White Screening Method for Selecting
a Clone (or Recombinant DNA Molecule)
Direct selection of engineered vector via antibioticresistance markers (ampR) on plasmid vectors.
Vector also contains-galactosidase gene for bluewhite 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
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.
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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)
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Transformation
Cloning genes
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 (RTPCR): Reverse transcriptase
makes DNA from viral RNA or
mRNA
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RT-PCR with a norovirus primer
Clinical Focus, p. 266
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?
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A Typical Genetic Modification Procedure
Foundation Figure
Fig 9.1
Fig 9.1
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A Typical Genetic Modification Procedure
Fig 9.1
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