Biotechnology
Chapter 17
DNA Manipulation
The molecular biology revolution started with
the discovery of restriction endonucleases
-Enzymes that cleave DNA at specific sites
These enzymes are significant in two ways
1. Allow a form of physical mapping that was
previously impossible
2. Allow the creation of recombinant DNA
molecules (from two different sources)
2
DNA Manipulation
Restriction enzymes recognize DNA
sequences termed restriction sites
There are two types of restriction enzymes:
-Type I = Cut near the restriction site
-Rarely used in DNA manipulation
-Type II = Cut at the restriction site
-The sites are palindromes
-Both strands have same sequence
3
when read 5’ to 3’
DNA Manipulation
Type II enzymes produce staggered cuts that
generate “sticky ends”
-Overhanging complementary ends
Therefore, fragments cut by the same enzyme
can be paired
DNA ligase can join the two fragments
forming a stable DNA molecule
4
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EcoRI
DNA
duplex
EcoRI
Restriction sites
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
A
Sticky ends
G
C
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
A
A
T
T
C
C
T
T
A
A
G
Recombinant DNA molecule
DNA ligase
joins the strands.
5
Gel Electrophoresis
A technique used to separate DNA fragments
by size
The gel (agarose or polyacrylamide) is
subjected to an electrical field
The DNA, which is negatively-charged,
migrates towards the positive pole
-The larger the DNA fragment, the slower it
will move through the gel matrix
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 of fragments
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
2
1
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.
Visualizing Stained Gel
Gel is stained with a dye to allow
the fragments to be visualized.
Longer
fragments
Shorter
fragments
c.
7
Transformation
Transformation is the introduction of DNA
from an outside source into a cell
Natural transformation occurs in many species
-However, not in E. coli, which is used
routinely in molecular biology labs
-Artificial transformation techniques have
been developed to introduce foreign
DNA into it
8
Molecular Cloning
A clone refers to a genetically identical copy
Molecular cloning is the isolation of a specific
DNA sequence (usually protein-encoding)
-Sometimes called gene cloning
The most flexible and common host for cloning
is E. coli
Propagation of DNA in a host cell requires a
vector
9
Vectors
Plasmids are small, circular
extrachromosomal DNA molecules
-Used for cloning small pieces of DNA
-Have three important components
1. Origin of replication
2. Selectable marker
3. Multiple cloning site (MCS)
10
Vectors
11
Vectors
Phage vectors are modified bacterial viruses
-Most based on phage lambda (l) of E. coli
-Used to clone inserts up to 40 Kbp
-Have two features not shared with plasmid
vectors
-They kill their host cells
-They have linear genomes
-Middle replaced with inserted DNA
12
Vectors
13
Vectors
Artificial chromosomes
-Used to clone very large DNA fragments
-Bacterial artificial chromosomes (BACs)
-Yeast artificial chromosomes (YACs)
14
DNA Libraries
A collection of DNA fragments from a specific
source that has been inserted into host cells
A genomic library represents the entire
genome
A cDNA library represents only the
expressed part of the genome
-Complementary DNA (cDNA) is
synthesized from isolated mRNA using the
enzyme reverse transcriptase
15
16
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exons
introns
1
1
2
2
3
3
Eukaryotic DNA template
Transcription
5´ cap
4
4
3´ poly-A tail
Primary RNA transcript
Introns are cut out,
and coding regions are
spliced together.
5´ cap
3´ poly-A tail
Mature RNA transcript
Isolation of mRNA
Addition of reverse
transcriptase Reverse
transcriptase
Reverse
transcriptase
utilizes mRNA
to create cDNA.
mRNA–cDNA hybrid
Addition of mRNAdegrading enzymes
Degraded
mRNA
DNA polymerase
Double-stranded cDNA
with no introns
17
DNA Libraries
Molecular hybridization is a technique used
to identify specific DNAs in complex mixtures
-A known single-stranded DNA or RNA is
labeled
-It is then used as a probe to identify its
complement via specific base-pairing
-Also termed annealing
18
DNA Libraries
Molecular hybridization is the most common
way of identifying a clone in a DNA library
-This process involves three steps:
1. Plating the library
2. Replicating the library
3. Screening the library
19
5. A comparison with the original plate
identifies the colony containing the
Filter paper
Film
1. Colonies of plasmid
containing bacteria, each
containing a single DNA
from the library, are grown
on agar.
2. A replica of the
4. The only sites on the
filter that will retain
probe DNA will contain
DNA complementary
to the probe. These
represent the sites of
colonies containing
the gene of interest.
plate is made by
pressing a piece of
filter paper against
the agar and
bacterial colonies.
Some cells from
each colony adhere
to the filter.
3. The filter is washed with a solution to break the cells
open and denature the DNA, which sticks to the filter at
the site of each colony. The filter is incubated with a
radioactively labeled probe that can form hybrids with
complementary DNA in the gene of interest.
20
DNA Analysis
Restriction maps
-Molecular biologists need maps to analyze
and compare cloned DNAs
-The first maps were restriction maps
-Initially, they were created by enzyme
digestion & analysis of resulting patterns
-Many are now generated by computer
searches for cleavage sites
21
DNA Analysis
Southern blotting
-A sample DNA is digested by restriction
enzymes & separated by gel electrophoresis
-Gel is transferred (“blotted”) onto a
nitrocellulose filter
-Then hybridized with a cloned,
radioactively-labeled DNA probe
-Complementary sequences are
revealed by autoradiography
22
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Electrophoretic
gel
2. The gel is covered
Stack of paper towels
with a sheet of
nitrocellulose and
placed in a tray of
Gel
buffer on top of a
Sponge
sponge. Alkaline
chemicals in the buffer
denature the DNA into
single strands. The
buffer wicks its way up
through the gel and
nitrocellulose into a
stack of paper towels
placed on top of the
nitrocellulose.
3. DNA in the gel is
transferred, or
“blotted,” onto the
nitrocellulose.
Gel
4. Nitrocellulose
with bound DNA
is incubated with
radioactively
labeled nucleic
acids and is
then rinsed.
Electrophoresis
1. Electrophoresis is Test nucleic
performed, using
radioactively labeled acids
markers as a size
guide in the first
Radioactively
lane.
labeled markers
with specific sizes
Nitrocellulose filter
Buffer
Nitrocellulose
paper now
contains nucleic
acid “print”
Sealed
container
Radioactive
probe (singlestranded DNA)
—AATGG—
—TTACC—
DNA fragments
within bands
5. Photographic
film is laid over
the filter and is
exposed only in
areas that contain
radioactivity
(autoradiography).
Bands on the film
represent DNA in
the gel that is
complementary to
the probe sequence.
Film
Hybridized nucleic
acids
Size markers
23
DNA Analysis
Northern blotting
-mRNA is electrophoresed and then blotted
onto the filter
Western blotting
-Proteins are electrophoresed and then
blotted onto the filter
-Detection requires an antibody that can
bind to one protein
24
DNA Analysis
RFLP analysis
-Restriction fragment length
polymorphisms (RFLPs) are generated by
point mutations or sequence duplications
-These fragments are often not identical in
different individuals
-Can be detected by Southern blotting
25
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Larger
fragments
restriction enzyme
cutting sites
Original Sequence
of Restriction Sites
(no mutations)
+
Point Mutations
Change the
Sequence of
Restriction Sites
Single base-pair
change
Sequence
Repetitions Can
Occur Between
Restriction Sites
Sequence duplication
+
a. Three different DNA
duplexes
b. Cut DNA
Smaller
fragments
–
+
–
+
–
+
c. Gel electrophoresis of
restriction fragments
26
DNA Analysis
DNA fingerprinting
-An identification technique used to detect
differences in the DNA of individuals
-Makes use of a variety of molecular
procedures, including RFLP analysis
-First used in a US criminal trial in 1987
-Tommie Lee Andrews was found guilty
of rape
27
DNA Analysis
28
DNA Analysis
DNA sequencing
-A set of nested
fragments is generated
-End with known base
-Separated by highresolution gel
electrophoresis,
resulting in a “ladder”
-Sequence is read from
the bottom up
29
DNA Analysis
DNA sequencing
-The enzymatic method
was developed by
Frederick Sanger
-Dideoxynucleotides
are used as chain
terminators in DNA
synthesis reactions
30
Manual Enzymatic DNA Sequencing
Template
DNA polymerase
3´
5´
T A G C C A T G C A
Primer
5´
5´
5´
A T C G
A T C G G
A T C G G T A C G
Reaction
for ddC
5´
A T C
5´
A T C G G T A C
Reaction
for ddA
5´
A
5´
A T C G G T A
5´
5´
5´
A T
A T C G G T
Reaction
for ddG
Reaction
for ddT
A T C G G T A C G T
G
C
A
T
3´
–
Longer
segments
T
G
C
A
T
G
G
C
Shorter
segments
+
T
A
5´
a.
31
DNA Analysis
DNA sequencing
-The enzymatic technique is powerful but is
labor intensive and time-consuming
-The development of automated techniques
made sequencing faster and more practical
-Fluorescent dyes are used instead of
radioactive labels
-Reaction is done in one tube
-Data are assembled by a computer
32
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Manual Enzymatic DNA Sequencing
Automated Enzymatic DNA Sequencing
Template
Template
DNA polymerase
3´
DNA polymerase
3´
5´
T A G C C A T G C A
5´
T A G C C A T G C A
Primer
Primer
5´
5´
5´
A T C G
A T C G G
A T C G G T A C G
5´
A
5´
A T
5´
A T C
Reaction
for ddC
5´
A T C
5´
A T C G
5´
A T C G G T A C
5´
A T C G G
Reaction
for ddA
5´
A
5´
A T C G G T
5´
A T C G G T A
5´
A T C G G T A
5´
5´
5´
A T
A T C G G T
5´
A T C G G T A C
5´
A T C G G T A C G
5´
A T C G G T A C G T
Reaction
for ddG
Reaction
for ddT
A T C G G T A C G T
G
C
A
T
Longer
segments
T 3´
G
C
A
T
G
G
C
T
A 5´
3´
–
T
G
Laser
C
Photo detector
reads colors
A
T
G
G
5´
A
C
Shorter
segments
+
C
G
G
T
A
C
G
T
3´
T
A
33
5´
a.
T
b.
DNA Analysis
Polymerase chain reaction (PCR)
-Developed by Kary Mullis
-Allows the amplification of a small DNA
fragment using primers that flank the region
-Each PCR cycle involves three steps:
1. Denaturation (high temperature)
2. Annealing of primers (low temperature)
3. DNA synthesis (intermediate temperature)
-Taq polymerase
34
DNA segment
to be amplified
5´
3´
3´
5´
PCR
machine
After 20 cycles, a
single fragment
produces over one
million (220) copies!
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´
3. DNA is heated to
72°C, the optimal
temperature for Taq
DNA polymerase to
extend primers.
3´
3´
5´
Taq DNA polymerase
5´
3´
3´
3´
5´
3´
3´
5´
5´
3´
3´
5´
5´
3´
3´
5´
5´
3´
3´
5´
3´
5´
5´
3´
5´
5´
5´
5´
3´
5´
Cycle 3:
8 copies
Cycle 2:
4 copies
5´
3´
5´
3´
3´
5´
5´
3´
3´
5´
5´
3´
3´
5´
5´
3´
3´
5´
3´
5´
5´
3´
3´
5´
35
DNA Analysis
Polymerase chain reaction (PCR)
-Has revolutionized science and medicine
because it allows the investigation of minute
samples of DNA
-Forensics
-Detection of genetic defects in embryos
-Analysis of mitochondrial DNA from
early human species
36
DNA Analysis
Yeast two-hybrid system
-Used to study protein-protein interactions
-Gal4 is a transcriptional activator with a
modular structure
-The Gal4 gene is split into two vectors
-Bait vector: has DNA-binding domain
-Prey vector: has transcription-activating
domain
-Neither of these alone can activate
37
transcription
DNA Analysis
Yeast two-hybrid system
-When other genes are inserted into these
vectors, they produce fusion proteins
-Contain part of Gal4 and the protein of
interest
-If the proteins being tested interact, Gal4
function will be restored
-A reporter gene will be expressed
-Detected by an enzyme assay 38
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Yeast nucleus
Transcriptionactivating domain
Yeast cell
Gal4 protein
DNAbinding
domain
DNA
DNAbinding
domain
Bait vector
RNA polymerase
Prey vector
Inserted DNA
Inserted DNA
Fusion
proteins
Prey protein
Bait protein
39
Reporter gene
Genetic Engineering
Has generated excitement and controversy
Expression vectors contain the sequences
necessary to express inserted DNA in a
specific cell type
Transgenic animals contain genes that have
been inserted without the use of
conventional breeding
40
Genetic Engineering
In vitro mutagenesis
-Ability to create mutations at any site in a
cloned gene
-Has been used to produce knockout mice,
in which a known gene is inactivated
-The effect of loss of this function is then
assessed on the entire organism
-An example of reverse genetics
41
42
Medical Applications
Human proteins
-Medically important proteins can be
produced in bacteria
-Human insulin
-Interferon
-Atrial peptides
-Tissue plasminogen activator
-Human growth hormone
43
Medical Applications
44
Medical Applications
Vaccines
-Subunit vaccines: Genes encoding a part
of the protein coat are spliced into a
fragment of the vaccinia (cowpox) genome
-DNA vaccines: Depend on the cellular
immune response (not antibodies)
45
Medical Applications
2. Herpes simplex
1. DNA is
extracted.
gene is isolated.
Gene specifying herpes
simplex surface protein
3. Vaccinia DNA
Herpes simplex virus
Human immune
response
is extracted
and cleaved.
Harmless vaccinia
(cowpox) virus
4. Fragment containing
surface gene
combines with
cleaved vaccinia DNA.
5. Harmless engineered
6. Antibodies directed
against herpes
simplex viral coat
are made.
virus (the vaccine)
with surface like
herpes simplex is
injected into the
human body.
46
Medical Applications
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
-Successful at first, but then patients
developed a rare leukemia
47
Agricultural Applications
Ti (tumor-inducing) plasmid is the most
used vector for plant genetic engineering
-Obtained from Agrobacterium tumefaciens,
which normally infects broadleaf plants
-However, bacterium does not infect cereals
such as corn, rice and wheat
48
Agricultural Applications
Gene of
interest
Plasmid
Agrobacterium
Plant nucleus
2. A gene of interest is
1. Plasmid is
removed and cut
open with
restriction
endonuclease.
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.
49
Agricultural Applications
Gene guns
-Uses bombardment with tiny gold particles
coated with DNA
-Possible for any species
-However, the copy number of inserted
genes cannot be controlled
50
Agricultural Applications
Herbicide resistance
-Broadleaf plants have
been engineered to be
resistant to the
herbicide glyphosate
-This allows for no-till
planting
51
Agricultural Applications
Pest resistance
-Insecticidal proteins have been transferred
into crop plants to make them pest-resistant
-Bt toxin from Bacillus thuringiensis
Golden rice
-Rice that has been genetically modified to
produce b-carotene (provitamin A)
-Converted in the body to vitamin A
52
Agricultural Applications
Daffodil
phytoene
synthase
gene (psy)
Bacterial
carotene
desaturase
gene (crtI)
Daffodil
lycopene
b-cyclase
gene (lcy)
Genes introduced
into rice genome
Rice
chromosome
psy
crtI
lcy
Phytoene
synthase
Carotene
desaturase
b-Cyclase
Expression
in endosperm
GGPP
Phytoene
Lycopene
b-Carotene
(Provitamin A)
53
Agricultural Applications
Adoption of genetically modified (GM) crops
has been resisted in some areas because
of questions about:
-Crop safety for human consumption
-Movement of genes into wild relatives
-Loss of biodiversity
54
Agricultural Applications
Biopharming
-Transgenic plants are used to produce
pharmaceuticals
-Human serum albumin
-Recombinant subunit vaccines
-Against Norwalk and rabies viruses
-Recombinant monoclonal antibodies
-Against tooth decay-causing bacteria
55
Agricultural Applications
Transgenic animal technology has not been
as successful as that in plants
-One interesting example is the EnviroPig
-Engineered to carry the gene for the
enzyme phytase
-Breaks down phosphorus in feed
-Reduces excretion of harmful
phosphates in the environment
56
Agricultural Applications
57