© 2013 Pearson Education, Inc.Lectures by Edward J. Zalisko
PowerPoint® Lectures forCampbell Essential Biology, Fifth Edition, and
Campbell Essential Biology with Physiology,
Fourth Edition
– Eric J. Simon, Jean L. Dickey, and Jane B. Reece
Chapter 12DNA Technology
Biology and Society: DNA, Guilt, and Innocence
• DNA profiling is the analysis of DNA samples that
can be used to determine whether the samples
come from the same individual.
• DNA profiling can therefore be used in courts to
indicate if someone is guilty of a crime.
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• DNA technology has led to other advances in the
– creation of genetically modified crops and
– identification and treatment of genetic diseases.
Biology and Society: DNA, Guilt, and Innocence
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RECOMBINANT DNA TECHNOLOGY
• Biotechnology
– is the manipulation of organisms or their
components to make useful products and
– has been used for thousands of years to
– make bread using yeast and
– selectively breed livestock for desired traits.
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• Biotechnology today means the use of DNA
technology, techniques for
– studying and manipulating genetic material,
– modifying specific genes, and
– moving genes between organisms.
RECOMBINANT DNA TECHNOLOGY
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• Recombinant DNA is constructed when scientists
combine pieces of DNA from two different sources
to form a single DNA molecule.
• Recombinant DNA technology is widely used in
genetic engineering, the direct manipulation of
genes for practical purposes.
RECOMBINANT DNA TECHNOLOGY
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Applications: From Humulin to Foods to “Pharm” Animals
• By transferring the gene for a desired protein into a
bacterium or yeast, proteins that are naturally
present in only small amounts can be produced in
large quantities.
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Making Humulin
• In 1982, the world’s first genetically engineered
pharmaceutical product was sold.
• Humulin, human insulin
– was produced by genetically modified bacteria and
– is used today by more than 4 million people with
diabetes.
• Today, humulin is continuously produced in
gigantic fermentation vats filled with a liquid culture
of bacteria.
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• DNA technology is used to produce medically
valuable molecules, including
– human growth hormone (HGH),
– the hormone erythropoietin (EPO), which
stimulates production of red blood cells, and
– vaccines, harmless variants or derivatives of a
pathogen used to prevent infectious diseases.
Making Humulin
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Genetically Modified (GM) Foods
• Today, DNA technology is quickly replacing
traditional breeding programs.
• Scientists have produced many types of
genetically modified (GM) organisms, organisms
that have acquired one or more genes by artificial
means.
• A transgenic organism contains a gene from
another organism, typically of another species.
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• In the United States today, roughly half of the corn
crop and more than three-quarters of the soybean
and cotton crops are genetically modified.
• Corn has been genetically modified to resist insect
infestation, attack by an insect called the European
corn borer.
Genetically Modified (GM) Foods
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• Strawberry plants produce bacterial proteins that
act as a natural antifreeze, protecting the plants
from cold weather.
• Potatoes and rice have been modified to produce
harmless proteins derived from the cholera
bacterium and may one day serve as edible
vaccines.
Genetically Modified (GM) Foods
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• ―Golden rice 2‖
– is a transgenic variety of rice that carries genes from daffodils and corn and
– could help prevent vitamin A deficiency and resulting blindness.
Genetically Modified (GM) Foods
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“Pharm” Animals
• A transgenic pig has been produced that carries a gene for human hemoglobin, which can be
– isolated and
– used in human blood transfusions.
• In 2006, genetically modified pigs carried roundworm genes that produce proteins that convert less healthy fatty acids to omega-3 fatty acids.
• However, unlike transgenic plants, no transgenic animals are yet sold as food.
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Recombinant DNA Techniques
• Bacteria are the workhorses of modern
biotechnology.
• To work with genes in the laboratory, biologists
often use bacterial plasmids, small, circular DNA
molecules that replicate separately from the larger
bacterial chromosome.
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• Plasmids
– can carry virtually any gene,
– can act as vectors, DNA carriers that move genes
from one cell to another, and
– are ideal for gene cloning, the production of
multiple identical copies of a gene-carrying piece of
DNA.
Recombinant DNA Techniques
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• Recombinant DNA techniques can help biologists
produce large quantities of a desired protein.
Recombinant DNA Techniques
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Figure 12.UN01DNA isolated from two sources and cut by samerestriction enzyme
Gene of interest(could be obtained froma library or synthesized)
RecombinantDNA
Plasmid(vector)
Transgenic organisms
Useful products
Figure 12.8
Plasmid
Bacterial cellIsolate plasmids.1 2
3
4
5
6
7
Cut both DNAs
with same
enzyme.
Isolate DNA.
Gene
of
interest
Other
genes
DNA fragments
from cell
DNA
Cell containing
the gene of interest
Mix the DNA fragments and join them together.
Gene of interest
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
Recombinant bacteria
Bacterial cloneClone the bacteria.
Find the clone with the gene of interest.
A protein is used to
dissolve blood clots
in heart attack
therapy.
A protein is used to prepare
“stone-washed” blue jeans.
Bacteria
produce
proteins,
which can be harvested
and used directly.
The gene
and protein
of interest
are isolated
from the
bacteria.
Genes may
be inserted
into other
organisms.
Some uses
of genes
Some uses
of proteins
A gene for pest
resistance is
inserted into
plants.
A gene is used to alter
bacteria for cleaning
up toxic waste.
8
Figure 12.8c
8
Protein for
dissolving
clots
Protein for
“stone-washing”
jeans
Harvested
proteins
may be
used
directly.
The gene
and protein
of interest
are isolated
from the
bacteria.
Genes may
be inserted
into other
organisms.
Some uses
of genes
Some uses
of proteins
Gene
for pest
resistance
Genes for
cleaning up
toxic waste
A Closer Look: Cutting and Pasting DNA with Restriction Enzymes
• Recombinant DNA is produced by combining two
ingredients:
1. a bacterial plasmid and
2. the gene of interest.
• To combine these ingredients, a piece of DNA
must be spliced into a plasmid.
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• This splicing process can be accomplished by
– using restriction enzymes, which cut DNA at
specific nucleotide sequences (restriction sites),
and
– producing pieces of DNA called restriction
fragments with ―sticky ends‖ important for joining
DNA from different sources.
A Closer Look: Cutting and Pasting DNA with Restriction Enzymes
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• DNA ligase connects the DNA pieces into
continuous strands by forming bonds between
adjacent nucleotides.
A Closer Look: Cutting and Pasting DNA with Restriction Enzymes
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Figure 12.9-4
Recognition site (recognition sequence)
for a restriction enzyme
Restriction
enzyme
DNA
DNA
ligase
Recombinant DNA molecule
A DNA fragment is added from
another source.
A restriction enzyme cuts the
DNA into fragments.
Fragments stick together by
base pairing.
DNA ligase joins the fragments
into strands.
1
2
3
4
A Closer Look: Obtaining the Gene of Interest
• How can a researcher obtain DNA that encodes a
particular gene of interest?
– A ―shotgun‖ approach can yield millions of
recombinant plasmids carrying many different
segments of foreign DNA.
– A collection of cloned DNA fragments that includes
an organism’s entire genome (a complete set of its
genes) is called a genomic library.
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• Once a genomic library is created, the bacterial
clone containing the desired gene is identified
using a nucleic acid probe consisting of a short
single strand of DNA with a complementary
sequence and labeled with either a radioactive
isotope or a fluorescent dye.
A Closer Look: Obtaining the Gene of Interest
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Figure 12.10
Radioactive probe(single-stranded DNA)
Single-stranded DNA
Mix with single-stranded DNA from various bacterial clones
Base pairing indicates the gene of interest
• Another way to obtain a gene of interest is to
– use reverse transcriptase and
– synthesize the gene by using an mRNA template.
A Closer Look: Obtaining the Gene of Interest
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Figure 12.11
Cell nucleus
DNA ofeukaryoticgene
Test tube
Transcription
Exon Intron Exon ExonIntron
RNA
transcript
mRNA
Introns removed
and exons spliced
together
Isolation of mRNA
from cell and
addition of
reverse transcriptase
Synthesis of cDNA
strand
Synthesis of second
DNA strand by DNA
polymerase
Reverse
transcriptase
cDNA strandbeing synthesized
cDNAof genewithoutintrons
2
3
1
5
4
• Another approach is to
– use an automated DNA-synthesizing machine and
– synthesize a gene of interest from scratch.
A Closer Look: Obtaining the Gene of Interest
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DNA PROFILING AND FORENSIC SCIENCE
• DNA profiling
– can be used to determine if two samples of genetic
material are from a particular individual and
– has rapidly revolutionized the field of forensics,
the scientific analysis of evidence from crime
scenes.
• To produce a DNA profile, scientists compare
sequences in the genome that vary from person to
person.
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Figure 12.UN02
Crime scene Suspect 1 Suspect 2
DNA
Polymerase chain
reaction (PCR)
amplifies STR
sites
Longer
DNA
fragments
Shorter
DNA
fragments
DNA fragments compared by gel electrophoresis
Gel
(Bands of shorter fragments move faster toward the positive pole.)
Investigating Murder, Paternity, and Ancient DNA
• DNA profiling can be used to
– test the guilt of suspected criminals,
– identify tissue samples of victims,
– resolve paternity cases,
– identify contraband animal products, and
– trace the evolutionary history of organisms.
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DNA Profiling Techniques The Polymerase Chain Reaction (PCR)
• The polymerase chain reaction (PCR)
– is a technique to copy quickly and precisely a
specific segment of DNA and
– can generate enough DNA, from even minute
amounts of blood or other tissue, to allow DNA
profiling.
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Short Tandem Repeat (STR) Analysis
• How do you test if two samples of DNA come from
the same person?
• Repetitive DNA
– makes up much of the DNA that lies between
genes in humans and
– consists of nucleotide sequences that are present
in multiple copies in the genome.
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• Short tandem repeats (STRs) are
– short sequences of DNA and
– repeated many times, tandemly (one after
another), in the genome.
• STR analysis
– is a method of DNA profiling and
– compares the lengths of STR sequences at
specific sites in the genome.
Short Tandem Repeat (STR) Analysis
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Figure 12.16
Crime scene DNA
Suspect’s DNA
Same number of
short tandem repeats
Different numbers of
short tandem repeats
STR site 1 STR site 2
AGAT
AGAT GATA
GATA
Gel Electrophoresis
• STR analysis
– compares the lengths of DNA fragments and
– uses gel electrophoresis, a method for sorting
macromolecules—usually proteins or nucleic
acids—primarily by their
– electrical charge and
– size.
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Figure 12.17-3
Band of longest
(slowest) fragments
Band of shortest
(fastest) fragments
Mixture of DNA
fragments of
different sizes
Power
source
• The DNA fragments are visualized as ―bands‖ on
the gel.
• The differences in the locations of the bands reflect
the different lengths of the DNA fragments.
Gel Electrophoresis
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• Gel electrophoresis may also be used for RFLP
analysis, in which DNA molecules are exposed to
a restriction enzyme, producing fragments that are
compared and made visible by gel electrophoresis.
RFLP Analysis
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Figure 12.19Crime scene DNA Suspect’s DNA
Fragment w
Fragment x
Fragment y
Longer
fragments
Shorter
fragments
Fragment z
Fragment y
Crime scene
DNA
Suspect’s
DNA
Cut
Cut Cut
Restriction
enzymes
added
x
wy y
z
GENOMICS AND PROTEOMICS
• Genomics is the study of complete sets of genes
(genomes).
– The first targets of genomics research were
bacteria.
– As of 2011,
– the genomes of more than 1,700 species have
been published and
– more than 8,000 are in progress.
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The Human Genome Project
• Begun in 1990, the Human Genome Project was a
massive scientific endeavor to
– determine the nucleotide sequence of all the DNA in
the human genome and
– identify the location and sequence of every gene.
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• At the completion of the project,
– more than 99% of the genome had been
determined to 99.999% accuracy,
– about 3 billion nucleotide pairs were identified,
– about 21,000 genes were found, and
– about 98% of the human DNA was identified as
noncoding.
The Human Genome Project
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• The Human Genome Project can help map the
genes for specific diseases such as
– Alzheimer’s disease and
– Parkinson’s disease.
The Human Genome Project
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Tracking the Anthrax Killer
• In October 2001,
– a Florida man died after inhaling anthrax and
– by the end of the year, four other people had also
died from anthrax.
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Tracking the Anthrax Killer
• In 2008, investigators
– completed a whole-genome analysis of the spores
used in the attack,
– found four unique mutations, and
– traced the mutations to a single flask at an Army
facility.
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Tracking the Anthrax Killer
• Although never charged, an army research scientist
suspected in the case committed suicide in 2008,
and the case remains officially unsolved.
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• The anthrax investigation is just one example of the new field of bioinformatics, the application of computational tools to molecular biology. Additional examples include
– evidence that a Florida dentist transmitted HIV to several patients,
– tracing the West Nile virus outbreak in 1999 to a single natural strain of virus infecting birds and people, and
– determining that our closest living relative, the chimpanzee (Pan troglodytes), shares 96% of our genome.
Tracking the Anthrax Killer
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Genome-Mapping Techniques
• Genomes are most often sequenced using the
whole-genome shotgun method, in which
– the entire genome is chopped into fragments using
restriction enzymes,
– all the fragments are cloned and sequenced, and
– computers running specialized mapping software
reassemble the millions of overlapping short
sequences into a single continuous sequence for
every chromosome—an entire genome.
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Figure 12.22-5
Chromosome
Chop up with
restriction enzyme
Sequence fragments
DNA fragments
Align fragments
Reassemble
full sequence
The Process of Science: Can Genomics Cure Cancer?
• Observation: A few patients responded quite
dramatically to a new drug, gefitinib, which
– targets a protein called EGFR found on the surface
of cells that line the lungs and
– is used to treat lung cancer.
• Question: Are genetic differences among lung
cancer patients responsible for the differences in
gefitinib’s effectiveness?
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• Hypothesis: Mutations in the EGFR gene were
causing the different responses to gefitinib.
• Prediction: DNA profiling that focuses on the
EGFR gene would reveal different DNA sequences
in the tumors of responsive patients compared with
the tumors of unresponsive patients.
The Process of Science: Can Genomics Cure Cancer?
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• Experiment: The EGFR gene was sequenced in
the cells extracted from the tumors of
– five patients who responded to the drug and
– four who did not.
The Process of Science: Can Genomics Cure Cancer?
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• Results: The results were quite striking.
– All five tumors from gefitinib-responsive patients
had mutations in EGFR.
– None of the other four tumors did.
– These results suggest that doctors can use DNA
profiling techniques to screen lung cancer patients
for those who are most likely to benefit from
treatment with this drug.
The Process of Science: Can Genomics Cure Cancer?
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Proteomics
• Success in genomics has given rise to
proteomics, the systematic study of the full set of
proteins found in organisms.
• To understand the functioning of cells and
organisms, scientists are studying
– when and where proteins are produced and
– how they interact.
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HUMAN GENE THERAPY
• Human gene therapy
– is a recombinant DNA procedure,
– seeks to treat disease by altering the genes of the
afflicted person, and
– often replaces or supplements the mutant version
of a gene with a properly functioning one.
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Figure 12.UN03
RNA versionof a normalhuman gene
Virus withRNA genome
Bonemarrow
A normal human gene is transcribedand translated in a patient, potentiallycuring the genetic disease permanently
Figure 12.24
Normal
human gene
Healthy person
The engineered
cells are injected
into the patient.
Bone of person
with diseaseBone
marrow
Bone marrow cell from the patient
Viral DNA carrying the human gene
inserts into the cell’s chromosome.
Bone marrow cells of the patient
are infected with the virus.
Inserted human RNA
RNA genome of virus
An RNA version of a normal human
gene is inserted into a harmless
RNA virus.
1
2
3
4
• Severe combined immunodeficiency (SCID) is
– a fatal inherited disease and
– caused by a single defective gene that prevents
the development of the immune system.
• SCID patients quickly die unless treated with
– a bone marrow transplant or
– gene therapy.
HUMAN GENE THERAPY
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• From 2000 to 2011, gene therapy has cured 22
children with inborn SCID.
• However, there have been some serious side
effects. Four of the children developed leukemia,
which proved fatal to one.
HUMAN GENE THERAPY
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SAFETY AND ETHICAL ISSUES
• As soon as scientists realized the power of DNA
technology, they began to worry about potential
dangers such as the
– creation of hazardous new pathogens and
– transfer of cancer genes into infectious bacteria
and viruses.
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SAFETY AND ETHICAL ISSUES
• Strict laboratory safety procedures have been
designed to
– protect researchers from infection by engineered
microbes and
– prevent microbes from accidentally leaving the
laboratory.
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The Controversy over Genetically Modified Foods
• GM strains account for a significant percentage of several staple crops in the United States.
• Advocates of a cautious approach are concerned that
– crops carrying genes from other species might harm the environment,
– GM foods could be hazardous to human health, and/or
– transgenic plants might pass their genes to close relatives in nearby wild areas.
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• Negotiators from 130 countries (including the
United States) agreed on a Biosafety Protocol that
– requires exporters to identify GM organisms
present in bulk food shipments and
– allows importing countries to decide whether the
shipments pose environmental or health risks.
The Controversy over Genetically Modified Foods
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• In the United States, all projects are evaluated for
potential risks by a number of regulatory agencies,
including the
– Food and Drug Administration,
– Environmental Protection Agency,
– National Institutes of Health, and
– Department of Agriculture.
The Controversy over Genetically Modified Foods
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Ethical Questions Raised by DNA Technology
• DNA technology raises legal and ethical questions—few of which have clear answers.
– Should genetically engineered human growth hormone be used to stimulate growth in HGH-deficient children?
– Should we try to eliminate genetic defects in our children and their descendants?
– Should people use mail-in kits that can tell healthy people their relative risk of developing various diseases?
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• DNA technologies raise many complex issues that
have no easy answers.
• We as a society and as individuals must become
educated about DNA technologies to address the
ethical questions raised by their use.
Ethical Questions Raised by DNA Technology
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Evolution Connection:The Y Chromosome as a Window on History
• Barring mutations, the human Y chromosome
passes essentially intact from father to son.
• By comparing Y DNA, researchers can learn about
the ancestry of human males.
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• DNA profiling of the Y chromosome has revealed
that
– nearly 16 million men currently living may be
descended from Genghis Khan,
– nearly 10% of Irish men were descendants of Niall
of the Nine Hostages, a warlord who lived during
the 1400s, and
– the Lemba people of southern Africa are
descended from ancient Jews.
Evolution Connection:The Y Chromosome as a Window on History
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