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BIOLOGY
CONCEPTS & CONNECTIONS
Fourth Edition
Neil A. Campbell • Jane B. Reece • Lawrence G. Mitchell • Martha R. Taylor
CHAPTER 12
DNA Technology and
the Human Genome
Modules 12.1 – 12.6
From PowerPoint® Lectures for Biology: Concepts & Connections
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
From E.Coli to a Map of Our Genes
• Research on E. coli revealed
that these bacteria have a
sexual mechanism that can
bring about the combining of
genes from two different cells
• This discovery led to the
development of recombinant DNA technology
– a set of techniques for combining genes from
different sources
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• DNA technology has many useful applications
– The Human Genome Project
– The production of vaccines, cancer drugs, and
pesticides
– Engineered
bacteria that
can clean up
toxic wastes
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BACTERIA AS TOOLS FOR MANIPULATING
DNA
12.1 In nature, bacteria can transfer DNA in three
ways
• Transformation, the taking
up of DNA from the fluid
surrounding the cell
DNA enters
cell
Fragment of
DNA from
another
bacterial cell
Bacterial chromosome
(DNA)
Figure 12.1A
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• Transduction, the
transfer of bacterial
genes by a phage
• Conjugation, the union
of cells and the DNA
transfer between them
Mating bridge
Phage
Fragment of
DNA from
another
bacterial cell
(former phage
host)
Sex pili
Donor cell
(“male”)
Figure 12.1B
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Figure 12.1C
Recipient cell
(“female”)
• The transferred DNA is then integrated into the
recipient cell’s chromosome
Donated DNA
Degraded DNA
Crossovers
Recipient cell’s
chromosome
Figure 12.1D
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Recombinant
chromosome
12.2 Bacterial plasmids can serve as carriers for
gene transfer
• An F factor is a DNA
segment in bacteria that
enables conjugation
and contains an origin
of replication
F factor (integrated)
Male (donor) cell
Origin of F replication
Bacterial chromosome
F factor starts
replication and
transfer of chromosome
Recipient cell
Only part of the
chromosome transfers
Figure 12.2A
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Recombination can occur
F factor (plasmid)
Male (donor)
cell
Bacterial
chromosome
F factor starts
replication and
transfer
• An F factor can exist as a
plasmid, a small circular
DNA molecule separate
from the bacterial
chromosome
Plasmids
Plasmid completes
transfer and
circularizes
Cell now male
Figure 12.2B, C
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12.3 Plasmids are used to customize bacteria: An
overview
• Plasmids are key tools for DNA technology
– Researchers use plasmids to insert genes into
bacteria
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1
Bacterium
Plasmid
isolated
2
DNA
isolated
3 Gene
Bacterial
chromosome
Cell containing gene
of interest
inserted
into plasmid
Plasmid
Gene of
interest
Recombinant DNA
(plasmid)
4
DNA
Plasmid put into
bacterial cell
Recombinant
bacterium
5 Cell multiplies with
gene of interest
Copies of gene
Gene for pest
resistance
inserted into
plants
Copies of protein
Clones of cell
Gene used to alter bacteria
for cleaning up toxic waste
Protein used to
make snow form
at higher
temperature
Protein used to dissolve blood
clots in heart attack therapy
Figure 12.3
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12.4 Enzymes are used to “cut and paste” DNA
• Restriction enzymes
cut DNA at specific
points
• DNA ligase “pastes”
the DNA fragments
together
Restriction enzyme
recognition sequence
1
DNA
Restriction enzyme
cuts the DNA into
fragments
2
Sticky end
Addition of a DNA
fragment from
another source
3
Two (or more)
fragments stick
together by
base-pairing
• The result is
recombinant DNA
4
DNA ligase
pastes the strand
5
Figure 12.4
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Recombinant DNA molecule
12.5 Genes can be cloned in recombinant plasmids:
A closer look
• Bacteria take the recombinant plasmids and
reproduce
• This clones the plasmids and the genes they
carry
– Products of the gene can then be harvested
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E. coli
1 Isolate DNA
Human cell
from two sources
2 Cut both
Plasmid
DNAs with
the same
restriction
enzyme
DNA
Gene V
Sticky ends
3 Mix the DNAs; they join
by base-pairing
4 Add DNA ligase
to bond the DNA covalently
Recombinant DNA
plasmid
Gene V
5 Put plasmid into bacterium
by transformation
6 Clone the bacterium
Bacterial clone carrying many
copies of the human gene
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Figure 12.5
12.6 Cloned genes can be stored in genomic
libraries
• Recombinant DNA
technology allows
the construction of
genomic libraries
Genome cut up
with restriction
enzyme
Recombinant
plasmid
OR
– Genomic libraries are
sets of DNA fragments
containing all of an
organism’s genes
• Copies of DNA fragments
can be stored in a cloned
bacterial plasmid or phage
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Bacterial
clone
Plasmid
library
Recombinant
phage DNA
Phage
clone
Phage
library
Figure 12.6
OTHER TOOLS OF DNA TECHNOLOGY
12.7 Reverse transcriptase helps make genes for
cloning
• Reverse transcriptase can be used to make
smaller cDNA libraries
– These contain only the genes that are
transcribed by a particular type of cell
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CELL NUCLEUS
DNA of
eukaryotic
gene
Exon Intron
Exon
Intron Exon
1 Transcription
RNA
transcript
2 RNA splicing
(removes introns)
mRNA
3 Isolation of mRNA
TEST TUBE
Reverse transcriptase
from cell and addition
of reverse transcriptase;
synthesis of DNA strand
4 Breakdown of RNA
cDNA strand
5 Synthesis of second
DNA strand
cDNA of gene
(no introns)
Figure 12.7
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12.8 Nucleic acid probes identify clones carrying
specific genes
• A nucleic acid probe can tag a desired gene in a
library
Radioactive
probe (DNA)
Mix with singlestranded DNA from
various bacterial
(or phage) clones
Single-stranded
DNA
Base pairing
indicates the
gene of interest
Figure 12.8A
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• DNA probes can
identify a bacterial
clone carrying a
specific gene
Bacterial colonies containing
cloned segments of foreign DNA
Radioactive DNA
1 Transfer
Solution
containing
probe
cells to
filter
Filter
paper
2 Treat cells
on filter to
separate
DNA
strands
3 Add probe
to filter
Probe
DNA
Gene of
interest
Single-stranded
DNA from cell
Hydrogen-bonding
4 Autoradiography
Colonies of living
cells containing
gene of interest
Developed film
5 Compare autoradiograph
with master plate
Master plate
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Figure 12.8B
12.9 Connection: DNA microarrays test for the
expression of many genes at once
• A labeled probe can
reveal patterns of
gene expression in
different kinds of
cells
• This technique may
revolutionize the
diagnosis and
treatment of cancer
cDNA
DNA of gene
DNA
microarray,
actual size
(6,400 genes)
Figure 12.9
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12.10 Gel electrophoresis sorts DNA molecules by
size
• Restriction fragments of DNA can be sorted by
size
Mixture of DNA
molecules of
different sizes
Longer
molecules
Power
source
Gel
Shorter
molecules
Glass
plates
Completed gel
Figure 12.10
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12.11 Restriction fragment analysis is a powerful
method that detects differences in DNA
sequences
• Scientists can
compare DNA
sequences of
different
individuals based
on the size of the
fragments
Allele 1
Allele 2
w
Cut
z
x
Cut
y
Figure 12.11A
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Cut
y
DNA from chromosomes
1
2
Longer
fragments
Shorter
fragments
Figure 12.11B
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
• Radioactive
probes are
also used to
make
comparisons
1
Restriction fragment
preparation
Restriction
fragments
2
Gel electrophoresis
3
Blotting
4
Radioactive probe
Filter paper
Radioactive, singlestranded DNA (probe)
Probe
5
Detection of radioactivity
(autoradiography)
Film
Figure 12.11C
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12.12 The PCR method is used to amplify DNA
sequences
• The
polymerase
chain reaction
(PCR) can
quickly clone a
small sample
of DNA in a
test tube
Initial
DNA
segment
1
2
4
Number of DNA molecules
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8
Figure 12.12
THE CHALLENGE OF THE HUMAN GENOME
12.13 Most of the human genome does not consist
of genes
• The 23 chromosomes in the haploid human
genome contain about 3 billion nucleotide pairs
– This DNA is believed to include about 35,000
genes and a huge amount of noncoding DNA
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
• Much of the
noncoding
DNA consists
of repetitive
nucleotide
sequences
– One example
includes
telomeres at
the end of the
chromosomes
Repeated unit
End of
DNA
molecule
NUCLEOTIDE SEQUENCE OF A HUMAN TELOMERE
Figure 12.13A
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• Barbara McClintock discovered that segments
of DNA called transposons can move about
within a cell’s genome
Figure 12.13B, C
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12.14 Connection: The Human Genome Project is
unlocking the secrets of our genes
• The Human Genome Project involves:
– genetic and physical
mapping of chromosomes
– DNA sequencing
– comparison of
human genes
with those of
other species
Figure 12.14
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OTHER APPLICATIONS OF DNA
TECHNOLOGY
12.15 Connection: DNA technology is used in courts
of law
• DNA fingerprinting can help solve crimes
Defendant’s
blood
Blood from
defendant’s
clothes
Victim’s
blood
Figure 12.15A, B
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12.16 Connection: Recombinant cells and
organisms can mass-produce gene products
• Recombinant cells and organisms are used to
manufacture useful proteins
Table 12.16
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• These sheep
carry a gene for a
human blood
protein that is a
potential
treatment for
cystic fibrosis
Figure 12.16
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12.17 Connection: DNA technology is changing the
pharmaceutical industry and medicine
• Hormones, cancer-fighting drugs, and new
vaccines are being produced using DNA
technology
– This lab equipment
is used to produce
a vaccine against
hepatitis B
Figure 12.17
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12.18 Connection: Genetically modified organisms
are transforming agriculture
• New genetic varieties of animals and plants are
being produced
– A plant with a new trait can be created using the
Ti plasmid
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Agrobacterium
tumefaciens
DNA containing
gene for desired trait
1
Ti
plasmid
T DNA
Insertion of
gene into plasmid
using restriction
enzyme and DNA
ligase
Plant cell
2
Recombinant
Ti plasmid
Restriction
site
Introduction
into plant
cells in
culture
3
Regeneration
of plant
T DNA
carrying
new gene
within plant
chromosome
Plant with
new trait
Figure 12.18A
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• “Golden rice” has been genetically modified to
contain beta-carotene
– This rice could help prevent vitamin A
deficiency
Figure 12.18B
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12.19 Connection: Gene therapy may someday
help treat a variety of diseases
• Techniques for
manipulating DNA have
potential for treating
disease by altering an
afflicted individual’s genes
Cloned gene (normal allele)
1 Insert
normal gene
into virus
Viral nucleic
acid
Retrovirus
2 Infect bone
marrow cell
with virus
– Progress is slow, however
3 Viral DNA
inserts into
chromosome
– There are also ethical
questions related to gene
therapy
Bone marrow
cell from patient
Bone
marrow
4 Inject cells
Figure 12.19
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into patient
RISKS AND ETHICAL QUESTIONS
12.20 Connection: Could GM organisms harm
human health or the environment?
• Genetic engineering involves
some risks
– Possible ecological damage
from pollen transfer between
GM and wild crops
– Pollen from a transgenic variety
of corn that contains a pesticide
may stunt or kill monarch
caterpillars
Figure 12.20A, B
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12.21 Connection: DNA technology raises
important ethical questions
• Our new genetic knowledge
will affect our lives in
many ways
• The deciphering of the
human genome, in
particular, raises
profound ethical issues
– Many scientists have
counseled that we
must use the
information wisely
Figure 12.21A-C
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