Transcript The Living World

The Living World
Fourth Edition
GEORGE B. JOHNSON
10
Gene Technology
PowerPoint® Lectures prepared by Johnny El-Rady
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10.1 A scientific Revolution
Genetic engineering is the process of moving genes
from one organism to another
Having a major impact on agriculture & medicine
Fig. 10.1
Producing insulin
Curing disease
Increasing yields
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10.2 Restriction Enzymes
Restriction enzymes bind to specific short sequences
(usually 4- to 6- bases long) on the DNA
The nucleotide sequence on both
DNA strands is identical when
read in opposite directions
GAATTC
CTTAAG
Most restriction enzymes cut the DNA in a
staggered fashion
This generates “sticky” ends
These ends can pair with any other DNA
fragment generated by the same enzyme
The pairing is aided by DNA ligase
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Fig. 10.2
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10.3 The Four Stages of a Genetic
Engineering Experiment
All gene transfer experiments share four distinct
stages
1.
2.
3.
4.
Cleaving DNA
Producing recombinant DNA
Cloning
Screening
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Fig. 10.3
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1. Cleaving the DNA
The large number of fragments produced are
separated by electrophoresis
Fragments
appear as
bands under
fluorescent light
Fig. 10.4
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2. Producing Recombinant DNA
Fragments of source DNA are inserted into vectors
Vectors are plasmids or viruses that carry foreign
DNA into the host cell
Vector DNA is cut with the same enzyme as the
source DNA, thus allowing the joining of the two
3. Cloning
Host cells are usually bacteria
As each bacterial cell reproduces, it forms a clone
of cells containing the fragment-bearing vector
Together all clones constitute a clone library
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4. Screening
A preliminary screen of the clone library eliminates
1. Clones without vectors
2. Clones with vectors that do not contain DNA
The vector employed usually has genes for
a. Antibiotic resistance
This eliminates the first type of clones because they
are sensitive to antibiotics
b. b-galactosidase
This eliminates the second type of clones based on
X-gal metabolism and color changes
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Fig. 10.5
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4. Screening
To find the gene of interest, the clone library is
screened by a process termed hybridization
The cloned genes form base pairs with
complementary sequences on another nucleic
acid, termed the probe
The bacterial colonies are first grown on agar
They are then transferred to a filter
The filter is treated with a radioactive probe
The filter is then subjected to
autoradiography
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Fig. 10.6
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10.4 Working with DNA
Key techniques used by today’s genetic engineers
include
PCR amplification
Used to increase the amounts of DNA
cDNA formation
Used to build genes from their mRNA
DNA fingerprinting
Used to identify particular individuals
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PCR Amplification
The polymerase chain reaction (PCR) requires
primers
Short single-stranded sequences complementary
to regions on either side of the DNA of interest
PCR consists of three basic steps
1. Denaturation
2. Primer annealing
3. Primer extension
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Target sequence
Fig. 10.7
Heat
Primers
1 Denaturation
Cool
Cycle
1
2 Annealing of primers
2 copies
DNA polymerase
Free nucleotides
3 Primer extension
Heat
Cycle
2
4 copies
Cool
Heat
8 copies
Cool
Cycle
3
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cDNA Formation
The primary mRNA transcript contains exons and
introns
The processed mRNA contains only exons
It is used as a template to create a single strand of
DNA termed complementary DNA (cDNA)
cDNA is then converted to a double-stranded
molecule
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Fig. 10.8
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DNA Fingerprinting
This is a process that is used to determine if two
DNA samples are from the same source
The DNA from the two sources is fragmented using
restriction enzymes
The fragments are separated using gel
electrophoresis
They are transferred to a filter
The filters are screened with radioactive
probes
Then subjected to autoradiography
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Fig. 10.9 Two of the DNA profiles that lead to conviction
First time DNA
profiles were used
in court of law
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10.5 Genetic Engineering
and Medicine
Genetic engineering has been used in many medical
applications
1. Production of proteins to treat illnesses
2. Creation of vaccines to combat infections
3. Replacement of defective genes
Gene therapy is discussed in Chapter 12
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Making “Magic Bullets”
In diabetes, the body is unable to control levels of
sugar in the blood because of lack of insulin
Diabetes can be cured if the body is supplied
with insulin
The gene
encoding insulin
has been
introduced into
bacteria
Fig. 10.1
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Making “Magic Bullets”
Other genetically engineered drugs include
Has only one
extra gene:
HGH
Anticoagulants
Used to treat heart
attack patients
Factor VIII
Used to treat
hemophilia
Human growth
hormone (HGH)
Used to treat
dwarfism
Fig. 10.10
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Piggyback Vaccines
Genetic engineering has also been used to create
subunit vaccines against viruses
A gene encoding a viral protein is put into the DNA
of a harmless virus and injected into the body
The viral protein will elicit antibody production in
the animal
A novel kind of vaccine was introduced in 1995
The DNA vaccine uses plasmid vectors
It elicits a cellular immune response,
rather than antibody production
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Fig. 10.11 Constructing a piggyback vaccine for the herpes simplex virus
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10.6 Genetic Engineering
of Farm Animals
In 1994, the recombinant hormone bovine
somatotropin (BST) became commercially available
Dairy farmers used BST as a supplement to
enhance milk production in cows
Consumers are concerned about the presence of the
hormone in milk served to children
This fear is unfounded
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Fig. 10.12 The production of BST through genetic engineering
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10.7 Genetic Engineering
of Crop Plants
Successful manipulation of the genes of crop plants
has improved the quality of these plants
Pest resistance
Leads to a reduction in the use of pesticides
Bt, a protein produced by soil bacteria, is harmful
to pests but not to humans
The Bt gene has been introduced into tomato
plants, among others
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10.7 Genetic Engineering
of Crop Plants
Glyphosateresistant plants
Herbicide resistance
Crop plants have
been created that
are resistant to
glyphosate
Fig. 10.14
Glyphosatesensitive plants
Petunias
Herbicide resistance offers two main advantages
1. Lowers the cost of producing crops
2. Reduces plowing and conserves the top soil
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10.7 Genetic Engineering
of Crop Plants
More Nutritious Crops
Worldwide, two major deficiencies are iron and
vitamin A
Deficiencies are especially severe in developing
countries where the major staple food is rice
Ingo Potrykus, a Swiss
bioengineer, developed
transgenic “golden” rice
to solve this problem
Fig. 10.15
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Fig. 10.15 Transgenic “golden” rice
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Potential Risks of Genetically Modified (GM) Crops
The promise of genetic engineering is very much in
evidence
However, it has generated considerable
controversy and protest
Are genetic engineers “playing God” by
tampering with the genetic material?
Two sets of risks need to be considered
1. Are GM foods safe to eat?
2. Are GM foods safe for the environment?
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Potential Risks of Genetically Modified (GM) Crops
1. Are GM foods safe to eat?
The herbicide glyphosate blocks the synthesis of
aromatic amino acids
Humans don’t make any aromatic amino acids,
so glyphosate doesn’t hurt us
However, gene modifications that render plants
resistant to glyphosate may introduce novel
proteins
Moreover, introduced proteins may cause
allergies in humans
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Potential Risks of Genetically Modified (GM) Crops
2. Are GM foods safe for the environment?
Three legitimate concerns are raised
1. Harm to other organisms
Will other organisms be harmed
unintentionally?
2. Resistance
Will pests become resistant to pesticides?
3. Gene flow
What if introduced genes will pass from GM
crops to their wild or weedy relatives?
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Potential Risks of Genetically Modified (GM) Crops
Should GM foods be labeled?
Every serious scientific
investigation has
concluded that GM
foods are safe
So there is no health
need for a GM label
However, people have a
right to know what is in
their food
So there may be a
need for label after all
Fig. 10.16
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