Biotechnology
1
Recombinant DNA
• Discovery of restriction enzymes
revolutionized molecular biology.
• Restriction enzymes cut DNA at specific
sites
– Used by bacteria against viruses
– Allow a form of gene mapping that was
previously impossible
– Allow the creation of recombinant DNA
molecules (from two different sources)
2
3 Types of Restriction Enzymes
•Type I and III cleave with less precision and
are not used in manipulating DNA
•Type II
– Recognize specific DNA sequences
– Cleave at specific site within sequence
– Can lead to “sticky ends” that can be joined
• Blunt ends can also be joined
3
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EcoRI
DNA
duplex
Restriction sites
EcoRI
G
A
A
T
T
C
G
A
A
T
T
C
C
T
T
A
A
G
C
T
T
A
A
G
EcoRI
Restriction endonuclease
cleaves the DNA
A
A
T
T
EcoRI
Restriction endonuclease
cleaves the DNA
C
G
G
C
Sticky ends
T
A
T
T
A
A
Sticky ends
G
C
A
T
A
A
T
T
C
G
DNA from another source cut with the
same restriction endonuclease is added.
A
A
T
T
C
G
G
C
A
T
A
T
T
A
T
A
C
G
Recombinant DNA molecule
DNA ligase
joins the strands.
4
DNA ligase
– Joins the two fragments forming a stable DNA
molecule
– Catalyzes formation of a phosphodiester bond
between adjacent pieces of DNA
– Same enzyme joins Okazaki fragments on
lagging strand in replication
5
Gel Electrophoresis
• Separate DNA fragments by size
• Gel made of agarose or polyacrylamide
• Submersed in a solution that can carry electric
current
• Negatively-charged DNA (due to phosphates),
migrates towards the positive pole
• Larger fragments move slower, smaller move
faster
• DNA is visualized using fluorescent dyes
6
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Restriction Enzyme Digestion
Gel Electrophoresis
DNA samples are cut with restriction enzymes in three
different reactions producing different patterns off ragments.
Samples from the restriction enzyme digests are introduced into the gel.
Electric current is applied causing fragments to migrate through the gel.
Restriction endonuclease
1 cut site
Reaction Reaction Reaction
1
2
3
Power
source
Reaction 1
Short segment
Long segment
Mixture of DNA
fragments of
different sizes in
solution placed at
the top of “lanes” in
the gel
Lane
Restriction endonuclease
2 cut site
–
Cathode
Reaction 2
Gel
Medium segment
Medium segment
Restriction
endonuclease 3
+
Reaction 3
Anode
Buffer
Long segment
a.
Short segment
b.
7
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Visualizing Stained Gel
Electrophoresis in the Laboratory
Gel is stained with a dye to allow
the fragments to be visualized.
Longer
fragments
Shorter
fragments
c.
d.
d: Courtesy of Biorad Laboratories
8
Transformation
• Introduction of foreign DNA from an
outside source into a cell
• Natural process in many species
• Transgenic organisms are all or part
transformed cells
9
Molecular Cloning
• Clone – genetically identical copy
• Molecular cloning – isolation of a specific
DNA sequence (usually protein-encoding
exon)
– Sometimes called gene cloning
• The most flexible and common host for
cloning is E. coli
– Vector – carries DNA in host and can
replicate in the host
– Each host–vector system has specific uses
10
Types of Vectors
• Plasmids
– Small, circular chromosomes
– Used for cloning small pieces of DNA
A Plasmid Vector
Restriction
endonuclease
Foreign
DNA
lacZ gene
Transform
No DNA
inserted
Medium contains
ampicillin and X-gal
Ampicillin
resistance
gene
Restriction enzymes
cuts within
the laxZ gene
Foreign DNA
and DNA ligase
are added
DNA
inserted
Active lacZ
gene produces
blue colonies
Inactive lacZ
gene produces
white colonies
Transform
11
Types of Vectors
• Artificial chromosomes
– Useful because plasmids have limited insert
size
– Yeast artificial chromosomes (YACs)
– Bacterial artificial chromosomes (BACs)
– Allow for larger insert for large-scale analysis
of genomes
12
Plant genetic engineering
Gene of
interest
Plasmid
Agrobacterium
Plant nucleus
1. Plasmid is
removed and cut open
with restriction
endonuclease.
2. A gene of interest is
isolated from the DNA
of another organism
and inserted into the
plasmid. The plasmid
is put back into the
Agrobacterium.
3. When used to infect plant cells,
Agrobacterium duplicates part
of the plasmid and transfers the
new gene into a chromosome of
the plant cell.
4. The plant cell divides, and each
daughter cell receives the new
gene. These cultured cells can
be used to grow a new plant
with the introduced gene.
13
Polymerase chain reaction (PCR)
• Allows the reproduction of a small DNA fragment
• Each PCR cycle involves three steps:
1. Denaturation (high temperature)
2. Annealing of primers (low temperature)
3. DNA synthesis (intermediate temperature)
14
DNA segment
to be amplified
5´
3´
3´
5´
PCR
machine
1. Sample is first heated
to denature DNA.
DNA is denatured
into single strands
5´
3´
3´
5´
2. DNA is cooled to a
lower temperature
to allow annealing
of primers.
5´
3´
Primers anneal to DNA
3´
5´
15
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After 20 cycles, a
single fragment
produces over one
million (220) copies!
3. DNA is heated to
72°C, the optimal
temperature for Taq
DNA polymerase to
extend primers
5´
3´
3´
5´
Taq DNA polymerase
3´
5´
3´
5´
3´
3´
5´
3´
5´
5´
3´
3´
5´
5´
3´
3´
5´
5´
3´
3´
5´
3´
5´
3´
5´
5´
5´
3´
Cycle 2:
4 copies
Cycle 3:
8 copies
5´
3´
3´
5´
3´
5´
3´
5´
3´
3´
5´
5´
3´
5´
5´
3´
3´
5´
3´
5´
5´
3´
3´
5´
16
• Applications of PCR
– Allows the investigation of minute samples of
DNA
– Forensics – drop of blood, cells at base of a
hair
– Detection of genetic defects in embryos by
analyzing a single cell
– Analysis of mitochondrial DNA from early
human species
17
DNA Fingerprinting
• RFLP analysis
– Restriction fragment length polymorphisms
– Generated by point mutations or sequence
duplications
– Restriction enzyme fragments are often not
identical in different individuals
18
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Original Sequence
of Restriction Sites
(no mutations)
Point Mutations
Change the
Sequence of
Restriction Sites
Sequence
Repetitions Can
Occur Between
Restriction Sites
Larger
fragments
restriction endonuclease
cutting sites
+
Single base-pair
change
Smaller
fragments
–
+
–
+
–
+
Sequence duplication
+
a. Three different
DNA duplexes
b. Cut DNA
c. Gel electrophoresis of
restriction fragments
19
• DNA fingerprinting
– Identification technique used to detect
differences in the DNA of individuals
– Short tandem repeats (STRs)
• Typically 2–4 nt long
• Not part of coding or regulatory regions
– Population is polymorphic for these markers
– Using several probes, probability of identity
can be calculated or identity can be ruled out
– Also used to identify remains
20
STR analysis
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STRs and DNA fingerprinting
1 Control Ladder
2
3
4
5
6
7
bp
DYS19
STR
Y chromosome
202 absent
198
194
190
186
178 absent
172
D12S66
STR
Chromosome 12
168
160
156
152 absent
148 absent
Data provided by Dr. L. Roewer, DNA Laboratory of the Institute for Forensic Medicine of Charité, Berlin
21
Medical Applications of
Biotechnology
• Medically important proteins can be
produced in bacteria
– Human insulin
– Vaccines
– Problem has been purification of desired
proteins from other bacterial proteins
22
Genetically engineering E. coli to make
human insulin
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In Humans
Promoter
Exon
Intron
In Bacterial Culture
Exon
Intron
Exon
AmpR
β-gal
Bacterial
promoter
Bacterial
promoter
Insulin A chain minus introns
and other “extra” sequence
Transcription
Exon
β-gal
Insulin B chain minus introns
and other “extra” sequence
Exon
Transform into E.coli
Translation
108 amino acids
Preproinsulin
Culture cells
Posttranslational modification
Cut
Purify β-gal-insulin
fusion proteins
Disulfide
bonds form
Cut
A chain
Purify A and Bchains
B chain
Cut
Achain
NH2
Bchain
NH2
Disulfide bond
Disulfide bond
a.
b.
COOH
COOH
Active
insulin
23
• Vaccines
– Subunit vaccines
• Genes encoding a part of the protein coat are
spliced into a fragment of the vaccinia (cowpox)
genome
• Injection of harmless recombinant virus leads to
immunity
– DNA vaccines
• Depend on the cellular immune response (not
antibodies)
24
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2. Herpes simplex
gene is isolated.
1. DNA is extracted.
3. Vaccinia DNA
is extracted and
cleaved.
Herpes simplex virus
Human immune
response
6. Antibodies directed
against herpes simplex
viral coat are made.
Gene specifying herpes
simplex surface protein
Harmless vaccinia
(cowpox) virus
4. Fragment containing
surface gene combines
with cleaved vaccinia DNA.
5. Harmless engineered
virus (the vaccine) with
surface like herpes
simplex is injected into
the human body .
25
• Gene therapy
– Adding a functional copy of a gene to correct
a hereditary disorder
– Severe combined immunodeficiency disease
(SCID) illustrates both the potential and the
problems
• On the positive side, 15 children treated
successfully are still alive
• On the negative side, three other children treated
have developed leukemia (due to therapy)
26
27
Agricultural Applications
• Herbicide resistance
– Broadleaf plants have been engineered to be
resistant to the herbicide glyphosate
– Benefits
• Crop resistant to glyphosate would not have to be
weeded
• Single herbicide instead of many types
• Glyphosate breaks down in environment
– In the United States, 90% of soy currently
grown is GM soy
28
• Bt crops
– Insecticidal proteins have been transferred
into crop plants to make them pest-resistant
– Bt toxin from Bacillus thuringiensis
– Use of Bt maize is the second most common
GM crop globally
• Stacked crops
– Both glyphosate-resistant and Bt-producing
29
• Golden rice
– Rice that has been genetically modified to
produce b-carotene (provitamin A)
– Converted in the body to vitamin A
– Interesting for 2 reasons
• Introduces a new biochemical pathway in tissue of
the transgenic plants
• Could not have been done by conventional
breeding as no rice cultivar known produces these
enzymes in endosperm
30
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Daffodil
phytoene
synthase
gene (psy )
Bacterial
carotene
desaturase
gene (crtI )
Daffodil
lycopene
β-cyclase
gene (lcy )
Genes introduced
Into rice genome
Rice
chromosome
psy
crtI
lcy
Expression
In endosperm
Phytoene
synthase
GGPP
β-Cyclase
Carotene
desaturase
Phytoene
Lycopene
β-Carotene
(Provitamin A)
31
Marker Assisted Breeding (MAB)
•
•
•
•
•
Combines classic plant breeding with molecular biology
DNA extracted from leaf tissue of young seedlings
Screen for agriculturally important traits
Screening uses DNA fingerprinting
Seedlings with desired traits raised to maturity
32
• Adoption of genetically modified (GM)
crops has been resisted in some areas
because of questions
– Crop safety for human consumption
– Movement of genes into wild relatives
• No evidence so far but it is not impossible
33
• Transgenic animal technology has not
been as successful as that in plants
• Molecular techniques combined with the
ability to clone domestic animals could
produce improved animals for
economically desirable traits
• Main use thus far has been engineering
animals to produce pharmaceuticals in
milk (biopharming)
34
Transgenic animals
Transgenic salmon
Wild salmon
6000
Weight (g)
5000
4000
3000
2000
1000
0
0
100
200 300
400 500
600 700
800 900
Days (from first feeding)
AquAdvantage salmon is a genetically modified (GM) Atlantic salmon
developed by AquaBounty Technologies. A growth hormone-regulating gene
from a Pacific Chinook salmon and a promoter from an ocean pout were added
to the Atlantic's 40,000 genes. These genes enable it to grow year-round instead
of only during spring and summer. The purpose of the modifications is to
increase the speed at which the fish grows, without affecting its ultimate size or
other qualities. The fish grows to market size in 16 to 18 months rather than
three years.