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Lectures by Chris C. Romero, updated by Edward J. Zalisko

PowerPoint

Lectures for
Campbell Essential Biology, Fourth Edition
Eric Simon, Jane Reece, and Jean Dickey
Campbell Essential Biology with Physiology, Third Edition
Eric Simon, Jane Reece, and Jean Dickey

Chapter 12
DNA 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
Innocent
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DNA technology has led to other advances in the:
Creation of genetically modified crops
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
Has been used for thousands of years to
Make bread using yeast
Selectively breed livestock for desired traits

Biotechnology today means the use of DNA technology, methods
for:
Studying and manipulating genetic material
Modifying specific genes
Moving genes between organisms

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Recombinant DNA is formed when scientists combine
nucleotide sequences (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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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 worlds first genetically engineered pharmaceutical
product was sold.
Humulin, human insulin:
Was produced by genetically modified bacteria
Was the first recombinant DNA drug approved by the FDA
Is used today by more than 4 million people with diabetes

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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 EPO, which stimulates production of red blood cells
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 plant-
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 one-half of the corn crop and
over three-quarters of the soybean and cotton crops are
genetically modified.
Corn has been genetically modified to resist insect infestation,
such as this damage caused by the European corn borer.
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Golden rice has been genetically modified to produce beta-
carotene used in our bodies to make vitamin A.
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Pharm Animals
In 2009 the FDA approved the first drug produced by livestock
that has been engineered to carry a human gene.
This product is a human anti-clotting protein collected from goats
milk.
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DNA technology:
May eventually replace traditional animal breeding but
Is not currently used to produce transgenic animals sold as food
Meat may come from livestock that receive genes that produce:
Larger muscles or
Healthy omega-3 fatty acids instead of less healthy fatty acids (already
done in 2006 in pigs)
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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 are
separate from the much larger bacterial chromosome.
Plasmids
Bacterial
chromosome
Remnant of
bacterium
C
o
l
o
r
i
z
e
d

T
E
M

Figure 12.7
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Plasmids:
Can easily incorporate foreign DNA
Are readily taken up by bacterial cells
Can act as vectors, DNA carriers that move genes from one cell to
another
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 can help biologists produce large
quantities of a desired protein.
Plasmid
Bacterial cell
Isolate
plasmids.
Figure 12.8-1
Plasmid
Bacterial cell
Isolate
plasmids.
DNA
Isolate
DNA.
Cell containing
the gene of interest
Figure 12.8-2
Plasmid
Bacterial cell
Isolate
plasmids.
DNA
Isolate
DNA.
DNA fragments
from cell
Cut both DNAs
with same
enzyme.
Gene of
interest
Other
genes
Cell containing
the gene of interest
Figure 12.8-3
Plasmid
Bacterial cell
Isolate
plasmids.
Gene of interest
Recombinant DNA plasmids
DNA
Isolate
DNA.
DNA fragments
from cell
Cut both DNAs
with same
enzyme.
Gene of
interest
Other
genes
Mix the DNAs and
join them together.
Cell containing
the gene of interest
Figure 12.8-4
Plasmid
Bacterial cell
Isolate
plasmids.
Recombinant bacteria
Gene of interest
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
DNA
Isolate
DNA.
DNA fragments
from cell
Cut both DNAs
with same
enzyme.
Gene of
interest
Other
genes
Mix the DNAs and
join them together.
Cell containing
the gene of interest
Figure 12.8-5
Plasmid
Bacterial cell
Isolate
plasmids.
Clone the bacteria.
Recombinant bacteria
Bacterial clone
Gene of interest
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
DNA
Isolate
DNA.
DNA fragments
from cell
Cut both DNAs
with same
enzyme.
Gene of
interest
Other
genes
Mix the DNAs and
join them together.
Cell containing
the gene of interest
Figure 12.8-6
Plasmid
Bacterial cell
Isolate
plasmids.
Find the clone with
the gene of interest.
Clone the bacteria.
Recombinant bacteria
Bacterial clone
Gene of interest
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
DNA
Isolate
DNA.
DNA fragments
from cell
Cut both DNAs
with same
enzyme.
Gene of
interest
Other
genes
Mix the DNAs and
join them together.
Cell containing
the gene of interest
Figure 12.8-7
Plasmid
Bacterial cell
Isolate
plasmids.
Some uses
of genes
Gene for pest
resistance
Gene for
toxic-cleanup
bacteria
Genes may be
inserted into
other organisms.
Find the clone with
the gene of interest.
The gene and protein
of interest are isolated
from the bacteria.
Clone the bacteria.
Recombinant bacteria
Bacterial clone
Gene of interest
Recombinant DNA plasmids
Bacteria take up recombinant plasmids.
Harvested
proteins may be
used directly.
Some uses
of proteins
Protein for
stone-washing
jeans
DNA
Cell containing
the gene of interest
Protein for
dissolving
clots
Isolate
DNA.
DNA fragments
from cell
Cut both DNAs
with same
enzyme.
Gene of
interest
Other
genes
Mix the DNAs and
join them together.
Figure 12.8-8
A Closer Look: Cutting and Pasting DNA with
Restriction Enzymes
Recombinant DNA is produced by combining two ingredients:
A bacterial plasmid
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, and
Producing pieces of DNA called restriction fragments with sticky
ends important for joining DNA from different sources
DNA ligase connects the DNA pieces into continuous strands by
forming bonds between adjacent nucleotides.
Recognition sequence
for a restriction enzyme
Restriction
enzyme
DNA
A restriction enzyme cuts
the DNA into fragments.
Figure 12.9-1
Recognition sequence
for a restriction enzyme
Restriction
enzyme
DNA
A DNA fragment is added
from another source.
A restriction enzyme cuts
the DNA into fragments.
Figure 12.9-2
Recognition sequence
for a restriction enzyme
Restriction
enzyme
DNA
A DNA fragment is added
from another source.
A restriction enzyme cuts
the DNA into fragments.
Fragments stick together by
base pairing.
Figure 12.9-3
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.
Figure 12.9-4
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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 yields millions of recombinant plasmids carrying
many different segments of foreign DNA.
A collection of cloned DNA fragments that includes an organisms 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 specific sequence of
radioactive nucleotides matching those in the desired gene, called
a nucleic acid probe.
Radioactive probe
(single-stranded DNA)
Single-stranded DNA
Mix with single-stranded DNA from
various bacterial clones
Base pairing indicates the
gene of interest
Figure 12.10
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Another way to obtain a gene of interest is to:
Use reverse transcriptase and
Synthesize the gene by using an mRNA template
Cell nucleus
DNA of
eukaryotic
gene
Test tube
Transcription
Exon Intron Exon Exon Intron
Figure 12.11-1
Cell nucleus
DNA of
eukaryotic
gene
RNA
transcript
mRNA
Test tube
Transcription
Introns removed and
exons spliced together
Exon Intron Exon Exon Intron
Figure 12.11-2
Cell nucleus
DNA of
eukaryotic
gene
RNA
transcript
mRNA
Test tube
Reverse
transcriptase
Transcription
Introns removed and
exons spliced together
Isolation of mRNA from
cell and addition of
reverse transcriptase
Exon Intron Exon Exon Intron
Figure 12.11-3
Cell nucleus
DNA of
eukaryotic
gene
RNA
transcript
mRNA
Test tube
Reverse
transcriptase
cDNA strand
being synthesized
Transcription
Introns removed and
exons spliced together
Isolation of mRNA from
cell and addition of
reverse transcriptase
Synthesis of cDNA
strand
Exon Intron Exon Exon Intron
Figure 12.11-4
Cell nucleus
DNA of
eukaryotic
gene
RNA
transcript
mRNA
Test tube
cDNA of gene
without introns
Reverse
transcriptase
cDNA strand
being synthesized
Transcription
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
Exon Intron Exon Exon Intron
Figure 12.11-5
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Another approach is to:
Use an automated DNA-synthesizing machine and
Synthesize a gene of interest from scratch

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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
Has rapidly revolutionized the field of forensics, the scientific analysis of
evidence from crime scenes
To produce a DNA profile, scientists compare genetic markers,
sequences in the genome that vary from person to person.
DNA isolated
Crime scene Suspect 1 Suspect 2
Figure 12.13-1
DNA isolated
DNA amplified
Crime scene Suspect 1 Suspect 2
Figure 12.13-2
DNA isolated
DNA amplified
DNA compared
Crime scene Suspect 1 Suspect 2
Figure 12.13-3
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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
Trace the evolutionary history of organisms
Figure 12.14
DNA Profiling Techniques
The Polymerase Chain Reaction (PCR)
The polymerase chain reaction (PCR):
Is a technique to copy quickly and precisely any 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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Initial
DNA
segment
Number of DNA molecules
1 2 4 8
Figure 12.15
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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
Repeated many times, tandemly (one after another), in the genome
STR analysis:
Is a method of DNA profiling
Compares the lengths of STR sequences at certain sites in the genome
Crime scene DNA
Suspects DNA
Same number of
short tandem repeats
Different numbers of
short tandem repeats
STR site 1 STR site 2
AGAT
AGAT GATA
GATA
Figure 12.16
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Gel Electrophoresis
STR analysis:
Compares the lengths of DNA fragments
Uses gel electrophoresis, a method for sorting macromoleculesusually
proteins or nucleic acidsprimarily by their
Electrical charge
Size
Mixture of DNA
fragments of
different sizes
Power
source
Gel
Figure 12.17-1
Mixture of DNA
fragments of
different sizes
Power
source
Gel
Figure 12.17-2
Mixture of DNA
fragments of
different sizes
Power
source
Gel
Completed gel
Band of
longest
(slowest)
fragments
Band of
shortest
(fastest)
fragments
Figure 12.17-3
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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.
Amplified
crime scene
DNA
Amplified
suspects
DNA
Longer
fragments
Shorter
fragments
Figure 12.18
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Gel electrophoresis may also be used for RFLP analysis, in
which DNA molecules are exposed to a restriction enzyme, which
produces fragments that are compared and made visible by gel
electrophoresis.
RFLP Analysis
Crime scene
DNA
Suspects
DNA
Fragment w
Fragment x
Fragment y
Longer
fragments
Shorter
fragments
Fragment z
Fragment y
Crime scene
DNA
Suspects
DNA
Cut
Cut Cut
Restriction enzymes added
x
w
y y
z
Figure 12.19
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GENOMICS AND PROTEOMICS
Genomics is the science of studying complete sets of genes
(genomes).
The first targets of genomics were bacteria.
As of 2009, the genomes of nearly one thousand species have been
published, including:
Bakers yeast
Mice
Fruit flies
Rice
Table 12.1
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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
To identify the location and sequence of every gene
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At the completion of the project in 2004:
Over 99% of the genome had been determined to 99.999% accuracy
3.2 billion nucleotide pairs were identified
About 21,000 genes were found
About 98% of the human DNA was identified as noncoding

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The Human Genome Project can help map the genes for specific
diseases such as:
Alzheimers disease
Parkinsons disease
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Tracking the Anthrax Killer
In October 2001:
A Florida man died after inhaling anthrax
By the end of the year, four other people had also died from anthrax
In 2008, investigators:
Completed a whole-genome analysis of the spores used in the attack
Found four unique mutations
Traced the mutations to a single flask at an Army facility
Anthrax
spore
Envelope
containing
anthrax spores
Figure 12.21
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The anthrax investigation is just one example of the new field of
comparative genomics, the comparison of whole genomes.
Comparative genomics has also provided strong evidence that:
A Florida dentist transmitted HIV to several patients
The West Nile virus outbreak in 1999 was a single natural strain of virus
infecting birds and humans
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
The fragments are cloned and sequenced
Computers running specialized mapping software reassemble the millions
of overlapping short sequences into a single continuous sequence for
every chromosomean entire genome
Chromosome
Figure 12.22-1
Chromosome
Chop up with
restriction enzyme
DNA fragments
Figure 12.22-2
Chromosome
Chop up with
restriction enzyme
Sequence
fragments
DNA fragments
Figure 12.22-3
Chromosome
Chop up with
restriction enzyme
Sequence
fragments
DNA fragments
Align
fragments
Figure 12.22-4
Chromosome
Chop up with
restriction enzyme
Sequence
fragments
DNA fragments
Align
fragments
Reassemble
full sequence
Figure 12.22-5
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Begun in 2006, the Human Variome Project:
Seeks to collect information on all of the genetic variations that affect
human health
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
Is used to treat lung cancer
Question: Are genetic differences among lung cancer patients
responsible for the differences in gefitinibs 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.
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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
Four who did not
Results: The results were quite striking.
All five tumors from gefitinib-responsive patients had mutations in
EGFR.
None of the other four tumors did.
Figure 12.23
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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
Often replaces or supplements the mutant version of a gene with a
properly functioning one
Normal human
gene isolated
and cloned
Healthy person
Figure 12.24-1
Normal human
gene isolated
and cloned
Normal human
gene inserted
into virus
Healthy person
Harmless
virus (vector)
Virus containing
normal human gene
Figure 12.24-2
Normal human
gene isolated
and cloned
Normal human
gene inserted
into virus
Virus injected
into patient with
abnormal gene
Healthy person
Harmless
virus (vector)
Virus containing
normal human gene
Bone
marrow
Bone of person
with disease
Figure 12.24-3
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SCID is a fatal inherited disease 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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Since the year 2000, gene therapy has:
Cured 22 children with inborn SCID but
Unfortunately, caused four of the patients to develop leukemia, killing
one of these children
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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
Transfer of cancer genes into infectious bacteria and viruses
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Strict laboratory safety procedures have been designed to:
Protect researchers from infection by engineered microbes
Prevent microbes from accidentally leaving the laboratory
Figure 12.25
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The Controversy over Genetically Modified
Foods
GM strains account for a significant percentage of several
agricultural crops in the United States.
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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
Transgenic plants might pass their genes to close relatives in nearby wild
areas
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In 2000, 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
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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
Department of Agriculture
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Ethical Questions Raised by DNA Technology
DNA technology raises legal and ethical questionsfew of which
have clear answers.
Should genetically engineered human growth hormone be used to
stimulate growth in HGH-deficient children?
Do we have any right to alter an organisms genesor to create new
organisms?
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?
Figure 12.27
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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.
Evolution Connection:
Profiling the Y Chromosome
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 10% of Irish men were descendants of Niall of the Nine Hostages,
a warlord who lived during the 5th century
The Lemba people of southern Africa are descended from ancient Jews
8% of males currently living in central Asia may be descended from
Genghis Khan