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CHAPTER
9
Biotechnology
and DNA
Technology
© 2016 Pearson Education, Inc.
© 2016 Pearson Education, Inc.
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.
© 2016 Pearson Education, Inc.
Introduction to Biotechnology
• Biotechnology: the use of microorganisms, cells,
or cell components to make a product
• Foods, antibiotics, vitamins, enzymes
• Recombinant DNA (rDNA) technology: the
insertion or modification of genes to produce
desired proteins
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An Overview of Recombinant DNA Procedures
• Vector: self-replicating DNA molecule used to
transport foreign DNA into a cell
• Clone: population of genetically identical cells
arising from one cell; each carries the vector
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Figure 9.1 A Typical Genetic Modification Procedure.
© 2016 Pearson Education, Inc.
Check Your Understanding
 Differentiate biotechnology and rDNA technology.
9-1
 In one sentence, describe how a vector and clone
are used.
9-2
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Tools of Biotechnology
Learning Objectives
9-3 Compare selection and mutation.
9-4 Define restriction enzymes, and outline how
they are used to make rDNA.
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.
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Tools of Biotechnology
• Selection: selecting for a naturally occurring
microbe that produces a desired product
• Mutation: Mutagens cause mutations that might
result in a microbe with a desirable trait
• Site-directed mutagenesis: a targeted and
specific change in a gene
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Restriction Enzymes
• Cut specific sequences of DNA
• Destroy bacteriophage DNA in bacterial cells
• Methylated cytosines in bacteria protect their own
DNA from digestion
• Create blunt ends or staggered cuts known as
sticky ends
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Recombinant DNA Technology
PLAY
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Animation: Recombinant DNA Technology
Table 9.1 Selected Restriction Enzymes Used in rDNA Technology
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Figure 9.2 A restriction enzyme's role in making rDNA.
Recognition sites
DNA
Cut
Cut
A restriction enzyme cuts (red arrows)
double-stranded DNA at its particular
recognition sites, shown in blue.
Cut
Cut
These cuts produce a DNA fragment with
two sticky ends.
Sticky end
DNA from another source,
perhaps a plasmid, cut with
the same restriction enzyme
When two such fragments of DNA cut
by the same restriction enzyme come
together, they can join by base pairing.
The joined fragments will usually form either a linear
molecule or a circular one, as shown here for a plasmid.
Other combinations of fragments can also occur.
The enzyme DNA ligase is used to unite the
backbones of the two DNA fragments,
producing a molecule of rDNA.
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rDNA
Vectors
•
•
•
•
Carry new DNA to desired cells
Must be able to self-replicate
Plasmids and viruses can be used as vectors
Shuttle vectors exist in several different species
and can move cloned sequences among various
organisms
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Figure 9.3 A plasmid used for cloning.
lacZ
ampR
HindIII
BamHI
EcoRI
pUC19
ori
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Check Your Understanding
 How are selection and mutation used in
biotechnology?
9-3
 What is the value of restriction enzymes in rDNA
technology?
9-4
 What criteria must a vector meet?
9-5
 Why is a vector used in rDNA technology?
9-6
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Polymerase Chain Reaction
• Process of increasing small quantities (amplifying)
of DNA for analysis
• Used for diagnostic tests for genetic diseases and
detecting pathogens
• Reverse-transcription PCR uses mRNA as
template
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PCR: Overview
PLAY
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Animation: PCR: Overview
PCR: Components
PLAY
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Animation: PCR: Components
Figure 9.4 The polymerase chain reaction.
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Check Your Understanding
 For what is each of the following used in PCR:
primer, DNA polymerase, 94C?
9-7
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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: Escherichia. coli, Saccharomyces
cerevisiae, mammalian cells, plant cells.
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Inserting Foreign DNA into Cells
• DNA can be inserted into a cell by:
• Transformation: Cells take up DNA from the
surrounding environment
• Electroporation: Electrical current forms pores in cell
membranes
• Protoplast fusion: Removing cell walls from two
bacteria allows them to fuse
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Figure 9.5 Protoplast fusion.
Chromosome
Plasma membrane
Cell wall
Bacterial cells
Bacterial cell walls
are enzymatically
digested, producing
protoplasts.
Protoplasts
In solution, protoplasts are
treated with polyethylene glycol.
Protoplasts fuse.
Segments of the two
chromosomes recombine.
Recombinant
cell
Recombinant cell
grows new cell wall.
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Inserting Foreign DNA into Cells
• DNA can be inserted into a cell by:
• Gene gun
• Microinjection
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Figure 9.6 A gene gun, which can be used to insert DNA-coated "bullets" into a cell.
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Figure 9.7 The microinjection of foreign DNA into an egg.
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Genomic Libraries
• Collections of clones containing different DNA
fragments
• An organism's DNA is digested and spliced into
plasmid or phage vectors and introduced into
bacteria
• At least one clone exists for every gene in the
organism
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Figure 9.8 Genomic libraries.
Genome to be stored
in library is cut up with
restriction enzyme
Recombinant
plasmid
OR
Host
cell
Recombinant
phage DNA
Phage cloning
vector
Plasmid Library
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Phage Library
Genomic Libraries
• Complementary DNA (cDNA) is made from
mRNA by reverse transcriptase
• Used for obtaining eukaryotic genes because
eukaryotic DNA has introns that do not code for
protein
• mRNA has the introns removed, coding only for
the protein product
© 2016 Pearson Education, Inc.
Figure 9.9 Making complementary DNA (cDNA) for a eukaryotic gene.
Exon Intron
Exon
Intron Exon
Nucleus
DNA
A gene composed of exons and
introns is transcribed to RNA by
RNA polymerase.
RNA
transcript
Processing enzymes in the nucleus
remove the intron-derived RNA
and splice together the
exon-derived RNA into mRNA.
mRNA
Cytoplasm
mRNA is isolated from the
cell, and reverse
transcriptase is added.
First strand
of DNA is
synthesized.
DNA strand
being synthesized
The mRNA is digested by
reverse transcriptase.
DNA polymerase is added to
synthesize second strand
of DNA.
cDNA of
gene without
introns
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Test tube
Synthetic DNA
• Builds genes using a DNA synthesis machine
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Figure 9.10 A DNA synthesis machine.
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Check Your Understanding
 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
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Selecting a Clone
• Blue-white screening
• Uses plasmid vector containing ampicillin resistance
gene (ampR) and β-galactosidase gene (lacZ)
• Bacteria is grown in media containing ampicillin and
X-gal, a substrate for β-galactosidase
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Figure 9.11 Blue-white screening, one method of selecting recombinant bacteria.
β-galactosidase gene (lacZ)
Ampicillin-resistance
gene (ampR)
Plasmid
Plasmid DNA and foreign DNA
are both cut with the same
restriction enzyme. The plasmid
has the genes for lactose hydrolysis
(the lacZ gene encodes the enzyme
β-galactosidase) and ampicillin
resistance.
Foreign DNA will insert into
the lacZ gene. The bacterium
receiving the plasmid vector
will not produce the enzyme
β-galactosidase if foreign
DNA has been inserted into
the plasmid.
The recombinant plasmid
is introduced into a
bacterium, which becomes
ampicillin resistant.
All treated bacteria are spread
on a nutrient agar plate
containing ampicillin and a
β-galactosidase substrate and
incubated. The β-galactosidase
substrate is called X-gal.
Only bacteria that picked up
the plasmid will grow in the
presence of ampicillin.
Bacteria that hydrolyze
X-gal produce galactose and
an indigo compound. The
indigo turns the colonies blue.
Bacteria that cannot hydrolyze
X-gal produce white colonies.
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Restriction
site
Foreign DNA
Restriction
sites
Recombinant
plasmid
Bacterium
Colonies
with foreign
DNA
Selecting a Clone
• Colony hybridization
• Use DNA probes: short segments of single-stranded
DNA complementary to the desired gene
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Figure 9.12 Colony hybridization: using a DNA probe to identify a cloned gene of interest.
Master plate with
colonies of bacteria
containing cloned
segments of foreign
genes
Nitrocellulose
filter
Replica plate
Make replica of master
plate on nitrocellulose
filter.
Treat filter with
detergent (SDS)
to lyse bacteria.
Strands of
bacterial DNA
Colonies containing
genes of interest
Compare filter with
replica of master plate
to identify colonies
containing gene of
interest.
Wash filter to
remove unbound
probe.
Bound
DNA
probe
Gene of
interest
Singlestranded
DNA
Treat filter with
sodium hydroxide
(NaOH) to separate
DNA into single
strands.
Probe will hybridize
with desired gene
from bacterial cells.
Fluorescence
labeled probes
Add labeled probes.
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Making a Gene Product
• E. coli
• Advantages: easily grown and its genomics are known
• Disadvantages: produces endotoxins and does not
secrete its protein products
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Figure 9.13 E. coli genetically modified to produce gamma interferon, a human protein that promotes an
immune response.
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Making a Gene Product
• Saccharomyces cerevisiae
• Easily grown and has a larger genome than bacteria
• Expresses eukaryotic genes easily
• Plant cells and whole plants
• Express eukaryotic genes easily
• Plants are easily grown, large-scale, and low-cost
• Mammalian cells
• Express eukaryotic genes easily
• Can make products for medical use
• Harder to grow
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Check Your Understanding
 How are recombinant clones identified?
9-11
 What types of cells are used for cloning rDNA?
9-12
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Applications of DNA Technology
Learning Objectives
9-13 List at least five applications of DN technology.
9-14 Define RNAi.
9-15 Discuss the value of genome projects.
9-16 Define the following terms: random shotgun
sequencing, bioinformatics, proteomics.
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Applications of DNA Technology
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.
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Therapeutic Applications
• Human enzymes and other proteins such as insulin
• Subunit vaccines: made from pathogen proteins
in genetically modified yeasts
• Nonpathogenic viruses carrying genes for
pathogen's antigens as DNA vaccines
• Gene therapy to replace defective or missing
genes
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Table 9.2 Some Pharmaceutical Products of rDNA (1 of 2)
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Table 9.2 Some Pharmaceutical Products of rDNA (2 of 2)
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Therapeutic Applications
• Gene silencing
• Small interfering RNAs (siRNAs) bind to mRNA,
which is then destroyed by RNA-induced silencing
complex (RISC)
• RNA interference (RNAi) inserts DNA encoding
siRNA into a plasmid and transferred into a cell
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Figure 9.14 Gene silencing could provide treatments for a wide range of diseases.
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Check Your Understanding
 Explain how DNA technology can be used to treat
disease and to prevent disease.
9-13
 What is gene silencing?
9-14
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Genome Projects
• Shotgun sequencing sequences small pieces of
genomes which are assembled by a computer
• Metagenomics is the study of genetic material
directly from environmental samples
• The Human Genome Project sequenced the entire
human genome
• The Human Proteome Project will map proteins
expressed in human cells
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Figure 9.15 Shotgun sequencing.
Isolate DNA.
Sequence DNA fragments.
Assemble sequences.
Fragment DNA
with restriction
enzymes.
Edit
sequences;
fill in gaps.
Clone DNA
in a bacterial
artificial
chromosome
(BAC).
Construct a gene library
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Random sequencing
Closure phase
Scientific Applications
• Bioinformatics: understanding gene function via
computer-assisted analysis
• Proteomics: determining proteins expressed in a
cell
• Reverse genetics: discovering gene function from
a genetic sequence
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Scientific Applications
• Southern blotting: DNA probes detect specific
DNA in fragments (RFLPs) separated by gel
electrophoresis
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Figure 9.16 Southern blotting.
Gel
Restriction enzyme
Larger
Gene of interest
Human
DNA
fragments
Smaller
The fragments are separated according to size by gel
electrophoresis. Each band contains many copies of a
particular DNA fragment. The bands are invisible but can
be made visible by staining.
DNA containing the gene of interest is extracted from
human cells and cut into fragments by restriction
enzymes. Fragments are called restriction fragment
length polymorphisms, or RFLPs (pronounced "rif-lips").
Paper towels
Nitrocellulose
filter
Salt solution
Sponge
Gel
Gel
Nitrocellulose
filter
The DNA bands are transferred to a nitrocellulose filter by
blotting. The solution passes through the gel and filter to
the paper towels by capillary action.
DNA
transferred
to filter
This produces a nitrocellulose filter with DNA fragments
positioned exactly as on the gel.
Labeled probes
Sealable
plastic bag
The filter is exposed to a labeled probe for a specific gene.
The probe will base-pair (hybridize) with a short sequence
present on the gene.
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The fragment containing the gene of interest is identified by a
band on the filter.
Forensic Microbiology
• DNA fingerprinting is used to identify pathogens
• PCR microarrays and DNA chips can screen
samples for multiple pathogens
• Differs from medicine because it requires:
• Proper collection of evidence
• Establishing a chain of custody
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Figure 9.17 DNA fingerprints used to track an infectious disease.
E. coli isolates from
patients whose
infections were
not juice related
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E. coli isolates from
patients who drank
contaminated juice
Apple juice
isolates
Nanotechnology
• Bacteria can make molecule-sized particles
• Nanospheres used in drug targeting and delivery
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Figure 9.18 Bacillus cells growing on selenium form chains of elemental selenium.
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Check Your Understanding
 How are shotgun sequencing, bioinformatics, and
proteomics related to genome projects?
9-15, 9-16
 What is Southern blotting?
9-17
 Why do RFLPs result in a DNA fingerprint?
9-18
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Agricultural Applications
• Ti plasmid: occurs in Agrobacterium tumefaciens
• Integrates into the plant genome and causes a tumorlike
growth
• Can be used to introduce rDNA into a plant
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Figure 9.19 Crown gall disease on a rose plant.
Crown gall
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Figure 9.20 Using the Ti plasmid as a vector for genetic modification in plants.
Agrobacterium tumefaciens
bacterium
Inserted T-DNA
carrying foreign
gene
The plasmid
is reinserted
into a bacterium.
Restriction
cleavage
site
Ti
plasmid
The bacterium is
used to insert the
T-DNA carrying the
foreign gene into the
chromosome of a
plant cell.
T-DNA
The plasmid is removed
from the bacterium, and
the T-DNA is cut by a
restriction enzyme.
Recombinant
Ti plasmid
The foreign DNA is
inserted into the T-DNA
of the plasmid.
Foreign DNA is cut
by the same enzyme.
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A plant is generated from a cell
clone. All of its cells carry the
foreign gene and may express
it as a new trait.
The plant cells
are grown in
culture.
Agricultural Applications
• Bt toxin
• Herbicide resistance
• Suppression of genes
• Antisense DNA
• Nutrition
• Human proteins
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Table 9.3 Some Agriculturally Important Products of rDNA Technology
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Check Your Understanding
 Of what value is the plant pathogen
Agrobacterium?
9-19
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Safety Issues and Ethics of Using DNA
Technology
Learning Objective
9-20 List the advantages of, and problems
associated with the use of genetic
modification techniques.
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Safety Issues and Ethics of Using DNA
Technology
• Need to avoid accidental release into the
environment
• Genetically modified crops must be safe for
consumption and for the environment
• Who will have access to an individual's genetic
information?
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Check Your Understanding
 Identify two advantages and two problems
associated with genetically modified organisms.
9-20
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Clinical Focus: Norovirus—Who Is Responsible
for the Outbreak?
• Are the outbreaks related?
• What is the source?
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Clinical Focus 9.1a
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Clinical Focus: Norovirus—Who Is Responsible
for the Outbreak?
• RT-PCR with a norovirus primer
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Clinical Focus 9.1b
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