Transcript 12_Lecture_Presentation

Chapter 12
DNA Technology
PowerPoint® Lectures for
Campbell Essential Biology, Fifth Edition, and
Campbell Essential Biology with Physiology,
Fourth Edition
– Eric J. Simon, Jean L. Dickey, and Jane B. Reece
Lectures by Edward J. Zalisko
© 2013 Pearson Education, Inc.
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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Figure 12.0
Biology and Society:
DNA, Guilt, and Innocence
• DNA technology has led to other advances in the
– creation of genetically modified crops and
– identification and treatment of genetic diseases.
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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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RECOMBINANT DNA TECHNOLOGY
• Biotechnology today means the use of DNA
technology, techniques for
– studying and manipulating genetic material,
– modifying specific genes, and
– moving genes between organisms.
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RECOMBINANT DNA TECHNOLOGY
• 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.
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Figure 12.1
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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Figure 12.2
Figure 12.3
Making Humulin
• 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.
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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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Genetically Modified (GM) Foods
• 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.
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Figure 12.4
Genetically Modified (GM) Foods
• 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.
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Genetically Modified (GM) Foods
• “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.
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Figure 12.5
“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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Figure 12.6
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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Figure 12.7
Bacterial
chromosome
Remnant of
bacterium
Colorized TEM
Plasmids
Recombinant DNA Techniques
• 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.
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Recombinant DNA Techniques
• Recombinant DNA techniques can help biologists
produce large quantities of a desired protein.
Animation: Cloning a Gene
Blast Animation: Genetic Recombination in Bacteria
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Figure 12.8
Bacterial cell
1
2 Isolate DNA.
Isolate plasmids.
Cell containing
the gene of interest
3
Plasmid
Cut both DNAs
with same
enzyme.
Gene
Other
of
interest genes
4
DNA fragments
from cell
DNA
Mix the DNA fragments and join them together.
Gene of interest
Recombinant DNA plasmids
5 Bacteria take up recombinant plasmids.
Recombinant bacteria
Bacterial clone
6 Clone the bacteria.
7 Find the clone with the gene of interest.
A gene for pest
resistance is
inserted into
plants.
Some uses
of proteins
Some uses
of genes
A gene is used to alter
bacteria for cleaning
up toxic waste.
8
Genes may
be inserted
into other
organisms.
The gene
and protein
of interest
are isolated
from the
bacteria.
Bacteria
produce
proteins,
which can be harvested
and used directly.
A protein is used to
dissolve blood clots
in heart attack
therapy.
A protein is used to prepare
“stone-washed” blue jeans.
Figure 12.8a
Bacterial cell
Cell containing
the gene of
interest
Plasmid
DNA
2 Isolate DNA.
1 Isolate plasmids.
3 Cut both DNAs
with same
enzyme.
Gene
Other
of
interest genes
DNA fragments
from cell
4 Mix the DNA fragments and join them together.
Gene of interest
Recombinant DNA plasmids
Figure 12.8b
5 Bacteria take up recombinant plasmids.
Recombinant bacteria
Bacterial clone
6 Clone the bacteria.
7 Find the clone with the gene of interest.
Figure 12.8c
Some uses
of genes
Genes for
cleaning up
toxic waste
Genes may
be inserted
into other
organisms.
Gene
for pest
resistance
Some uses
of proteins
8 The gene
and protein
of interest
are isolated
from the
bacteria.
Harvested
proteins
may be
used
directly.
Protein for
dissolving
clots
Protein for
“stone-washing”
jeans
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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A Closer Look: Cutting and Pasting DNA with
Restriction Enzymes
• 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.
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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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A Closer Look: Obtaining the Gene of Interest
• 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.
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Figure 12.10
Radioactive probe
(single-stranded DNA)
Mix with single-stranded DNA
from various bacterial clones
Single-stranded DNA
Base pairing indicates
the gene of interest
A Closer Look: Obtaining 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.
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Figure 12.11
Cell nucleus
Exon Intron Exon Intron Exon
DNA of
eukaryotic
gene
1 Transcription
RNA
transcript
mRNA
2 Introns removed
and exons spliced
together
Test tube
3 Isolation of mRNA
from cell and
addition of
reverse transcriptase
Reverse
transcriptase
cDNA strand
being synthesized
cDNA
of gene
without
introns
4 Synthesis of cDNA
strand
5 Synthesis of second
DNA strand by DNA
polymerase
A Closer Look: Obtaining the Gene of Interest
• Another approach is to
– use an automated DNA-synthesizing machine and
– synthesize a gene of interest from scratch.
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Figure 12.12
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.
Video: Biotechnology Lab
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Figure 12.13-1
1 DNA isolated
Crime scene Suspect 1 Suspect 2
Figure 12.13-2
1 DNA isolated
2 DNA amplified
Crime scene Suspect 1 Suspect 2
Figure 12.13-3
1 DNA isolated
2 DNA amplified
3 DNA compared
Crime scene Suspect 1 Suspect 2
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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Figure 12.14
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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Figure 12.15
Initial
DNA
segment
1
2
4
8
Number of DNA molecules
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 Repeat (STR) Analysis
• 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.
Blast Animation: DNA Fingerprinting
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Figure 12.16
Crime scene DNA
STR site 2
STR site 1
AGAT
Same number of
short tandem repeats
AGAT
Suspect’s DNA
GATA
Different numbers of
short tandem repeats
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.
Blast Animation: Gel Electrophoresis
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Figure 12.17-3
Mixture of DNA
fragments of
different sizes
Band of longest
(slowest) fragments
Power
source
Band of shortest
(fastest) fragments
Gel Electrophoresis
• 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.
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Figure 12.18
Amplified
crime scene
DNA
Amplified
suspect’s
DNA
Longer
fragments
Shorter
fragments
RFLP Analysis
• 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.
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Figure 12.19
Crime scene DNA
Suspect’s DNA
Fragment w
Cut
Fragment z
Restriction
enzymes
added
Fragment x
Cut
Cut
Fragment y
Fragment y
Crime scene
DNA
Longer
fragments
Suspect’s
DNA
z
x
Shorter
fragments
w
y
y
Figure 12.19a
Crime scene DNA
Suspect’s DNA
Fragment w
Cut
Fragment z
Restriction
enzymes
added
Fragment x
Cut
Fragment y
Cut
Fragment y
Figure 12.19b
Crime scene
DNA
Longer
fragments
Suspect’s
DNA
z
x
Shorter
fragments
w
y
y
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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Table 12.1
Table 12.1a
Table 12.1b
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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The Human Genome Project
• 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.
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The Human Genome Project
• The Human Genome Project can help map the
genes for specific diseases such as
– Alzheimer’s disease and
– Parkinson’s disease.
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Figure 12.20
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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Figure 12.21
Envelope
containing
anthrax spores
Colorized SEM
Anthrax
spore
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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Tracking the Anthrax Killer
• 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.
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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
DNA fragments
Sequence fragments
Align fragments
Reassemble
full sequence
Figure 12.22a
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 12.24
Normal
human gene
1 An RNA version of a normal human
gene is inserted into a harmless
RNA virus.
RNA genome of virus
Inserted human RNA
Healthy person
2
Bone marrow cells of the patient
are infected with the virus.
3 Viral DNA carrying the human gene
inserts into the cell’s chromosome.
Bone marrow cell from the patient
Bone
marrow
4 The engineered
cells are injected
into the patient.
Bone of person
with disease
HUMAN GENE THERAPY
• 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.
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HUMAN GENE THERAPY
• 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.
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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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Figure 12.25
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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Figure 12.26
The Controversy over Genetically Modified Foods
• 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.
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The Controversy over Genetically Modified Foods
• 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.
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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 HGHdeficient 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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Figure 12.27
Ethical Questions Raised by DNA Technology
• 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.
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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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Evolution Connection:
The Y Chromosome as a Window on History
• 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.
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Figure 12.28
Figure 12.UN01
DNA isolated from
two sources and
cut by same
restriction enzyme
Gene of interest
(could be obtained from
a library or synthesized)
Plasmid
(vector)
Recombinant
DNA
Transgenic organisms
Useful products
Figure 12.UN02
Crime scene
Suspect 1
Suspect 2
DNA
Polymerase chain
reaction (PCR)
amplifies STR
sites
Longer
DNA
fragments
Gel
Shorter
DNA
fragments
DNA fragments compared by gel electrophoresis
(Bands of shorter fragments move faster toward the positive pole.)
Figure 12.UN03
RNA version
of a normal
human gene
Virus with
RNA genome
Bone
marrow
A normal human gene is transcribed
and translated in a patient, potentially
curing the genetic disease permanently