Download 12.3 How Is Biotechnology Used In Forensic Science?

Chapter 12
Biotechnology
Lectures by
Gregory Ahearn
University of North Florida
Copyright © 2009 Pearson Education, Inc..
12.1 What Is Biotechnology?
 Biotechnology is any use or alteration of organisms,
cells, or biological molecules to achieve specific
practical goals.
• Today, selective ___________ is an important part of
biotechnology.
• Modern biotechnology frequently uses _________
engineering.
• This term refers to direct methods for modifying genetic
material.
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12.1 What Is Biotechnology?
 A key tool in genetic engineering is _____________ DNA,
which is DNA that has been altered to contain genes or
parts of genes from different organisms.
• Large amounts of recombinant DNA can be grown in
bacteria, viruses, or yeasts, and then transferred into other
species.
• Plants or animals that express DNA that has been
modified or derived from other species are called
______________, or genetically modified, organisms
(______s).
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12.1 What Is Biotechnology?
 Modern biotechnology also includes many methods of
manipulating DNA, whether or not the DNA is
subsequently put into a cell or an organism.
 For example, determining the nucleotide sequence of
specific pieces of DNA is crucial to forensic science
and the diagnosis of inherited disorders.
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12.1 What Is Biotechnology?

This chapter is organized around five themes.
1. Recombinant DNA mechanisms found in nature
1. Biotechnology in criminal forensics (DNA matching)
1. Biotechnology in agriculture, and production of transgenic
plants and animals
1. The Human Genome Project
1. Biotechnology in medicine; the treatment of inherited
disorders
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12.2 How Does DNA Recombine In Nature?
 Many natural processes can transfer DNA from one
organism to another, sometimes even to organisms of
different species.
• Sexual reproduction recombines DNA from two different
organisms.
• Every egg and sperm contain recombinant DNA, derived
from the organism’s two parents.
• When a sperm fertilizes an egg, the resulting offspring also
contains recombinant DNA.
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12.2 How Does DNA Recombine In Nature?
 Bacteria can undergo several
types of recombination that
allow gene transfer between
species.
bacterial
chromosome
plasmid
1 micrometer
(a) Bacterium
 In a process called
transformation, bacteria pick
up pieces of DNA from the
environment.
 The DNA may be part of the
chromosome of another
bacterium or from another
species, or may be in the form
of tiny circular DNA molecules
called ____________.
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bacterial
chromosome
bacterial
chromosome
plasmid
DNA
fragments
The plasmid replicates
in the cytoplasm
(c) Transformation with a plasmid
A DNA fragment is
incorporated into
the chromosome
(b) Transformation with a DNA fragment
Fig. 12-1
12.2 How Does DNA Recombine In Nature?
 Viruses may transfer DNA between species.
• Viruses are genetic material encased in a _________ coat,
and they transfer their genetic material to the host cells that
they infect.
• Once inside a host cell, the viral genes replicate and use
the infected cell’s enzymes and ribosomes to
synthesize viral proteins.
• These proteins form new viruses that are released to infect
other cells.
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12.2 How Does DNA Recombine In Nature?
 Viruses may transfer genes between cells.
virus
viral DNA
The virus enters
host
the host cell
cell
host cell DNA
A virus attaches to
a susceptible host cell
The virus releases its
DNA into the host cell;
some viral DNA (red) may
be incorporated into the
host cell’s DNA (blue)
viral DNA
“hybrid virus”
The host cell
bursts open, releasing
newly assembled
viruses; if “hybrid
viruses” infect a
second cell, they may
transfer genes from
the first cell to the
second cell
viral proteins
New viruses
assemble; some host
cell DNA is carried by
“hybrid viruses”
Viral genes encode
the synthesis of viral
proteins and viral gene
replication; some host
cell DNA may attach to
the replicated viral DNA
(red/blue combination)
Fig. 12-2
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12.3 How Is Biotechnology Used In
Forensic Science?
 The polymerase chain reaction (PCR) amplifies DNA
• PCR produces virtually unlimited amounts of selected pieces
of DNA.
• _________ (small pieces of complementary RNA) tell the
DNA polymerase where on the DNA molecule to start
copying.
• One primer is complementary to the beginning of the
DNA strand to be copied.
• The other primer is complementary to the other end, so
DNA replication occurs in both directions.
• PCR consists of the following steps repeated as often as
needed to make enough copies of DNA.
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12.3 How Is Biotechnology Used In
Forensic Science?

In a small test tube, DNA is mixed with primers, free
nucleotides, and a special heat-resistant DNA
polymerase.
1.
2.
3.
4.
The test tube is heated to 90°C, which breaks the
_________ bonds between complementary bases,
separating the DNA into single strands.
The temperature is lowered to about 50°C to allow the
primers to form complementary base pairs with the
original DNA strands.
The temperature is raised to 70–72°C so DNA polymerase
can use the primers to make copies of the DNA segment
bounded by the primers.
The cycle is repeated as many times as desired.
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12.3 How Is Biotechnology Used In
Forensic Science?
 PCR copies a specific DNA sequence.
194°F (90°C)122°F (50°C)
primers
original
DNA
161°F (72°C)
DNA
polymerase
Heating
Cooling allows
separates
primers and
DNA strands DNA polymerase
to bind
(a) One PCR cycle
new DNA
strands
New DNA
strands a
synthesized
Fig. 12-3a
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12.3 How Is Biotechnology Used In
Forensic Science?
DNA
fragment
to be
amplified
PCR
cycles
1
2
3
4 etc.
DNA
copies 1
2
4
8
16 etc.
(b) Each PCR cycle doubles the number of copies of the DNA
Fig. 12-3b
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12.3 How Is Biotechnology Used In
Forensic Science?
 Differences in ______ ________ repeats can identify
individuals.
• In many criminal investigations, PCR is used to amplify the
DNA so that there is enough to compare the DNA left at the
crime scene with the suspect’s DNA.
• Forensic experts have found that small segments of DNA,
called short tandem repeats (STRs), can be used to identify
people with astonishing accuracy.
• People have different numbers of repeated nucleotides in their STRs.
• A perfect match of 10 STRs in a suspect’s DNA and the DNA
found at a crime scene means that there is less than one chance
in a trillion that the two DNA samples did not come from the
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Pearson Education
Inc.
person.
12.3 How Is Biotechnology Used In
Forensic Science?
 Short tandem repeats
A T A T T T T G AA G A T A G A T A G A T A G A T A G A T A G A T A G A T A G A T A G G T A
T A T A AA A C T T C T AT C T AT C T A T C T AT C T A T C T AT C T A T C T A T C C A T
Eight side-by-side (tandem) repeats
of the same four-nucleotide sequence
AG A T
TC TA
Fig. 12-4
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12.3 How Is Biotechnology Used In
Forensic Science?
 Gel electrophoresis separates DNA segments.
• A mixture of DNA pieces is separated by a technique called
gel electrophoresis.
• The mixture of DNA is loaded onto a slab of _________.
• The gel is put in a chamber with __________ connected
to each end; one is positive, the other negative.
• Current is allowed to flow between the electrodes through
the gel.
• The flowing current separates the DNA fragments,
forming distinct bands on the gel.
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12.3 How Is Biotechnology Used In
Forensic Science?
 Gel electrophoresis
DNA samples are pipetted
into wells (shallow slots) in the
gel. Electrical current is sent
through the gel (negative at the
end with the wells, positive at
the opposite end).
power supply
pipetter
gel
wells
Fig. 12-5(1)
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12.3 How Is Biotechnology Used In
Forensic Science?
Electrical current moves the
DNA segments through the gel.
Smaller pieces of DNA move
farther toward the positive
electrode.
DNA “bands”
(not yet
visible)
Fig. 12-5(2)
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12.3 How Is Biotechnology Used In
Forensic Science?
 DNA probes are used to label specific nucleotide sequences.
• Unfortunately, the DNA bands are invisible, so how can anyone
identify which band contains a specific STR?
• Usually, the two strands of the DNA double helix are separated
during gel electrophoresis.
• This allows pieces of synthetic DNA, called DNA probes, to basepair with specific DNA fragments in the sample.
 The DNA probes are labeled, either by radioactivity or by
attaching colored molecules to them.
 A given DNA probe will only label certain DNA sequences and
not others.
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12.3 How Is Biotechnology Used In
Forensic Science?
 DNA probes identify
specific DNA
sequences.
probe
label
(colored molecule)
TC T ATCT ATC T A
T T T GA AG A T AG A T AG A T
STR #1: The probe base-pairs and binds
to the DNA
TC TATCT ATC T A
AC T GAAT G A A T G A A T G A A T G
STR #2: The probe cannot base-pair with the DNA,
so it does not bind
Fig. 12-6
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12.3 How Is Biotechnology Used In
Forensic Science?
 The single-stranded DNA segments are transferred out of the
gel and onto a piece of nylon paper.
The gel is placed on special
nylon “paper.” Electrical current
drives the DNA out of the gel
onto the nylon.
gel
nylon
paper
 The paper is next bathed in a solution containing a specific
DNA probe that will base-pair with, and bind to, only a specific
STR, making this STR visible.
Fig. 12-5(3)
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12.3 How Is Biotechnology Used In
Forensic Science?
The nylon paper with the
DNA bound to it is bathed in a
solution of labeled DNA probes
(red) that are complementary to
specific DNA segments in the
original DNA sample.
solution of
DNA probes
(red)
nylon
paper
Fig. 12-5(4)
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12.3 How Is Biotechnology Used In
Forensic Science?
Complementary DNA
segments are labeled by the
probes (red bands).
Fig. 12-5(5)
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12.3 How Is Biotechnology Used In
Forensic Science?
 Every person has a unique DNA profile.
• In the early 1990s, forensic scientists ran DNA samples from
a crime scene and from the suspects side-by-side on a gel,
to see which suspect’s DNA matched that found at the
scene.
• In modern STR analysis, a suspect and crime scene DNA
samples can be run on different gels in different locations.
• The reason is that people have different numbers of repeats
of their STRs, so that every person on Earth—except for
identical twins—has a unique set of STRs.
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12.3 How Is Biotechnology Used In
Forensic Science?
STR
name
Penta D
CSF
D16
Number of repeats
 When DNA samples are run on STR gels, they produce
a pattern, called a DNA profile
D16: An STR on chromosome 16
15
14
13
12
11
10
9
8
DNA samples from
13 different people
D7
Fig. 12-7
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12.3 How Is Biotechnology Used In
Forensic Science?
• Anyone convicted of certain crimes must give a blood
sample.
• Forensic technicians then determine the criminal’s DNA
profile.
• This DNA profile is coded and stored in computer files.
• Because all forensic labs use the same STRs,
computers can easily determine if DNA left at a crime
scene matches one of the millions of profiles stored in a
profile database.
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12.4 How Is Biotechnology Used In
Agriculture?
 Many crops are genetically modified.
• Many herbicides kill plants by inhibiting an enzyme that is
used by plants, but not animals, to synthesize essential
amino acids.
• Many herbicide-resistant transgenic crops have been given a
bacterial gene encoding an enyzme that functions even in the
presence of these herbicides.
• These plants continue to synthesize normal amounts of amino
acid and proteins.
• Less competition from weeds—which are killed by the
herbicide, but not the crops—provides larger harvests.
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12.4 How Is Biotechnology Used In
Agriculture?
• Insect resistance of many crops is enhanced by giving
them a gene, called Bt, from the bacterium, Bacillus
thuringiensis.
• The protein encoded by the Bt gene damages the
_________ tract of insects, but not mammals.
• Bt crops therefore suffer less damage from insects, and
farmers have to apply less pesticide to their fields.
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12.4 How Is Biotechnology Used In Agriculture?

How would a seed company make insect-resistant Bt plants?
1.
The desired gene is cloned.
1.
First, one must obtain the gene; then, it must be inserted into a
plasmid so that huge numbers of copies can be made.
1.
Restriction enzymes cut the DNA at specific nucleotide
sequences.
1.
Genes are inserted into plasmids through the action of
restriction enzymes isolated from bacteria.
1.
Each restriction enzyme cuts DNA at a specific nucleotide
sequence.
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12.4 How Is Biotechnology Used In Agriculture?
EcoRI restriction enzyme
doublestranded
DNA
. . .AA T T G C T TAG A AT T C G A T T T G. . .
. . . T T AA C G A AT C T T A A G C T A AA C . . .
A specific restriction enzyme (EcoRI) binds
to the GAATTC sequence and cuts the DNA,
creating DNA fragments with “sticky ends.”
. . .A A T T G C T T A G
. . .T TA A C GA AT C T TAA
A A T T C G A T T T G. . .
G C T A A A C. . .
single-stranded
“sticky ends”
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Fig. 12-8
12.4 How Is Biotechnology Used In Agriculture?
6.
Cutting two pieces of DNA with the same restriction enzyme allows
the pieces to be joined together.
DNA including Bt gene (blue)
plasmid
The DNA containing the Bt gene and the
plasmid are cut with the same restriction enzyme.
6.
The Bt gene is inserted into a plasmid by cutting the DNA on either
side of the Bt gene and splitting open the circle of the plasmid with
the same restriction enzyme.
Fig. 12-9
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12.4 How Is Biotechnology Used In Agriculture?
8.
As a result, the ends of the Bt gene and the opened-up plasmid
both have complementary bases in their ends, and can base-pair
with each other.
recombinant
plasmid with
Bt gene
Bt genes and plasmids, both with the same
complementary sticky ends, are mixed together;
DNA ligase bonds the Bt genes into the plasmids.
8.
When the cut Bt genes and plasmids are mixed together, some of
the Bt genes will be temporarily inserted between the ends of the
plasmid.
10. Adding DNA ligase permanently bonds the Bt genes into the
plasmid.
Fig. 12-9(2)
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12.4 How Is Biotechnology Used In Agriculture?
11. Plasmid-transformed bacteria insert the Bt gene into a plant.
12. Certain bacteria can enter the cells of specific types of plants.
13. These bacteria are transformed with the recombinant plasmids.
bacterium
bacterial
chromosome
recombinant
plasmids
Bacteria are transformed with the recombinant plasmids.
11. When the transformed bacteria enter a plant cell, the plasmids
insert their DNA, including the Bt gene, into the plant’s
chromosomes.
12. Therefore, any time that a plant cell divides, all of its daughter cells
inherit the Bt gene.
Fig. 12-9(3)
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12.4 How Is Biotechnology Used In Agriculture?
15. Hormones stimulate the transgenic plant cells to divide and
differentiate into entire plants.
plant chromosome
plant cell
Bt gene
Transgenic bacteria enter the plant cells, and Bt genes
are inserted into the chromosomes of the plant cells.
15. These plants are bred to one another, or to other plants, to create
commercially valuable plants that resist insect attack.
Fig. 12-9(4)
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12.4 How Is Biotechnology Used In Agriculture?
 Bt plants resist insect attack.
Fig. 12-10
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12.4 How Is Biotechnology Used In Agriculture?
 Genetically modified plants may produce medicines.
• Similar techniques can be used to insert medically useful genes
into plants, producing medicines.
• Plants could be made to produce human antibodies that would
combat various diseases.
• A direct injection of plant-produced antibodies soon after infection
might cure the resulting disease much more rapidly than
waiting for the immune system to handle the pathogens.
• Plant-derived antibodies against bacteria that cause tooth decay
and non-Hodgkins lymphoma could be produced cheaply,
enhancing the availability of therapies.
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12.4 How Is Biotechnology Used In Agriculture?
 Genetically modified animals may be useful in agriculture
and medicine.
• Making transgenic animals involves injecting the desired DNA into
a fertilized egg, which is then implanted into a surrogate mother.
• If the offspring are healthy and express the foreign gene, they are then
bred together to produce homozygous transgenic organisms.
• Companies have developed salmon and trout with modified or added
growth-hormone genes, which make the fish grow much faster than wild
fish.
• Because medicines are generally more valuable than meat, many
researchers are developing animals that will produce medicines.
• Alpha-1-antitrypsin: a protein that may prove value in treating cystic fibrosis
• Erythropoietin: a hormone that stimulates red blood cell production
• Clotting factors for treating hemophilia
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12.5 How Is Biotechnology Used To Learn About
The Human Genome?
 In 1990, the Human Genome Project was launched to
determine the nucleotide sequence of all DNA in our entire
set of genes, called the human genome.
• The human genome contains only about 21,000 genes comprising
only 2% of the DNA.
• It is not known what the 98% of the remaining DNA does.
 Knowing the human genome will help:
•
•
•
•
Find out what many unknown proteins do.
Understand human disease.
Diagnose genetic disorders and devise treatments or cures.
Comparing the human genome to that of other organisms will help
us understand our place in the evolution of life.
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12.6 How Is Biotechnology Used For Medical
Diagnosis And Treatment?
 DNA technology can be used to diagnose inherited
disorders.
• People inherit a genetic disease because they inherit one or
more dysfunctional alleles.
• Defective alleles have different nucleotide sequences than
functional alleles.
• Two methods are currently used to find out if a person
carries a normal allele or a malfunctioning allele.
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12.6 How Is Biotechnology Used For Medical
Diagnosis And Treatment?
 Restriction enzymes may cut different alleles at different locations.
• A defective allele may be cut by a particular restriction enzyme, while a
functional allele will not.
MstII
cut #1
MstII
cut #2
MstII
cut #3
DNA probe
large piece
small piece
of DNA
of DNA
(a) MstII cuts the normal globin allele into two pieces
that can be labeled by a probe
Fig. 12-11a
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12.6 How Is Biotechnology Used For Medical
Diagnosis And Treatment?
MstII
cut #1
MstII
cut #3
DNA probe
very large piece
of DNA
(b)MstII cuts the sickle-cell allele into one very large
piece that can be labeled by the same probe
Fig. 12-11b
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12.6 How Is Biotechnology Used For Medical
Diagnosis And Treatment?
larger pieces
of DNA
smaller pieces
of DNA
Homozygous normal:
one band of large DNA
pieces and one band
of small DNA pieces
Homozygous
sickle-cell: one
band of very
large DNA
pieces
Heterozygous:
three bands
(c) Analysis of globin alleles by gel electrophoresis
Fig. 12-11c
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12.6 How Is Biotechnology Used For Medical
Diagnosis And Treatment?
 Different alleles bind to different DNA probes.
• This method has been applied to characterizing the 1,000 different CFTR
(cystic fibrosis) protein alleles.
DNA probe
for normal
CFTR allele
DNA probes for
10 different mutant
CFTR alleles
(a) Linear array of probes for cystic fibrosis
colored molecule
A T C A T C T T T GG T G
piece of patient’s
DNA
(b) CFTR allele labeled with a colored
molecule
Fig. 12-12a,b
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12.6 How Is Biotechnology Used For
Medical Diagnosis And Treatment?
#1
Homozygous for normal CFTR alleles—
the person is phenotypically normal
#2
One normal and one defective CFTR allele—
the person is phenotypically normal
#3
Two different defective CFTR alleles—
the person develops cystic fibrosis
(c) Linear arrays with labeled DNA samples
from three different people
Fig. 12-12c
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12.6 How Is Biotechnology Used For Medical
Diagnosis And Treatment?
 Someday, physicians may be able to use an
array containing hundreds or thousands
of DNA probes for hundreds of diseaserelated alleles.
 This will help them determine the
susceptibility for the diseases that each
patient has, and to tailor the patient’s
medical care accordingly.
 Arrays containing probes for thousands of
human genes are already manufactured.
 These arrays can be tailored to investigate
gene activity in specific diseases, such as
breast cancer.
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Fig. 12-13
12.6 How Is Biotechnology Used For Medical
Diagnosis And Treatment?
 DNA technology can be used to treat disease.
• Thanks to recombinant DNA technology, several medically important
proteins are now routinely made in bacteria.
• These proteins may prevent or cure a variety of diseases, but they
cannot cure inherited disorders—they only treat symptoms.
 Biotechnology may be able to treat inherited disorders in the future.
• Patients with defective CFTR proteins in their lung cells can have viruses
containing normal CFTR alleles sprayed into their nose.
• The viruses enter the lung cells, and the normal CFTR allele directs the
synthesis of normal CFTR protein.
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12.6 How Is Biotechnology Used For Medical
Diagnosis And Treatment?
 Using biotechnology to cure severe combined immune deficiency
(SCID)
• In the body, new cells are produced from stem cells that can
differentiate into several possible cell types.
• SCID is a rare, inherited disorder in which a child fails to develop an
immune system; most die before their first birthday.
• This disorder is due to a defective allele that does not produce an
enzyme called adenosine deaminase.
• A 4-year old patient had her white blood cells treated with a virus
containing a functional allele, and these cells were returned to her body.
• Now an adult, she has a normally functioning immune system but must
receive continual injections of new treated white cells, since the original
treated white cells have died off.
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12.7 What Are Some Of The Major Ethical
Issues Of Modern Biotechnology?
 Should genetically modified organisms (GMOs) be permitted in
agriculture?
• Even though transgenic crops offer clear advantages to farmers, many
people strenuously object to transgenic crops or livestock.
• They fear that they may be hazardous to human health or dangerous
to the environment.
• In most cases, there is no reason to think that GMO foods are
dangerous to eat.
• The Bt protein is not toxic to mammals, and should not prove a
danger to human health.
• However, some people might be allergic to genetically modified plants.
• A plant made transgenic by the incorporation of a new gene may confer
upon the crop a trait that induces allergic reactions in some people.
• In 2003, the U.S. Society of Toxicology studied the risks of genetically
modified plants and concluded that the current transgenic plants pose
no dangers to human health.
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12.7 What Are Some Of The Major Ethical
Issues Of Modern Biotechnology?
 Are GMOs hazardous to the environment?
• Bt genes used in rice fields to add herbicide resistance to the rice
may escape in the pollen and enter plants far away from the
rice it was intended to control.
• The plants receiving the unintentional genes may then also
obtain herbicide resistance.
• If these plants are weeds, they will become resistant to
control by the herbicides and will negatively affect the rice
crops.
• In 2002, a committee of the U.S. National Academy of Sciences
recommended more thorough screening of transgenic plants
before they are used commercially, and sustained ecological
monitoring after commercialization
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12.7 What Are Some Of The Major Ethical
Issues Of Modern Biotechnology?
 What about transgenic animals?
• Some transgenic animals, such as fish, have the
potential to pose significant threats to the environment.
• If they escaped, they might be more aggressive, grow faster,
or mature faster than wild fish, and might replace native
populations.
• Commercial fish farms market only sterile transgenic salmon
and trout so that escapees would die without
reproducing, and thus have minimal effect on the
environment.
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parents with genetic disease
 Should the genome of
humans be changed by
biotechnology?
• Should people be
allowed to select, or
even change, the
genomes of their
offspring?
fertilized
egg with a
defective
gene
embryo
with a
genetic
defect
baby with
a genetic
disorder
treated culture
therapeutic
gene
viral
vector
Fig. 12-14
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 If it were possible to insert alleles
encoding functional CFTR
proteins into human eggs, thereby
preventing cystic fibrosis, would
this be an ethical change to the
human genome?
genetically corrected
cell from culture
 What about making a bigger and
stronger football player?
 When the technology is
developed to cure diseases, it will
be difficult to prevent it from being
used for nonmedical purposes.
therapeutic
gene
egg cell
without
a nucleus
genetically
corrected
egg cell
genetically
corrected
clone of the
original embryo
healthy baby
Fig. 12-14
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