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Figure 20.1
Chapter 20
Biotechnology
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
The DNA Toolbox
• Sequencing of the genomes of more than 7,000
species was under way in 2010
• Recombinant DNA  nucleotide sequences from
two different sources combined in vitro into the
same DNA molecule
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2011 Pearson
Education, Inc.
• Genetic engineering  direct manipulation of
genes for practical purposes
• Biotechnology  manipulation of organisms or
their genetic components to make useful products
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2011 Pearson
Education, Inc.
DNA Cloning
• Foreign DNA inserted into plasmid*  plasmid
inserted into bacterial cell
• Reproduction in bacterial cell  cloning of
plasmid with foreign DNA
•  multiple copies of a single gene
*Plasmids are small circular DNA molecules that replicate separately
from the bacterial chromosome
© 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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2011 Pearson
Education, Inc.
Figure 20.2
Bacterium
1 Gene inserted into
plasmid
Bacterial
Plasmid
chromosome
Recombinant
DNA (plasmid)
Cell containing gene
of interest
Gene of
interest
2 Plasmid put into
bacterial cell
DNA of
chromosome
(“foreign” DNA)
Recombinant
bacterium
3 Host cell grown in culture to
form a clone of cells containing
the “cloned” gene of interest
Protein expressed from
gene of interest
Gene of
interest
Protein harvested
Copies of gene
Basic
research
on gene
4 Basic research
and various
applications
Basic
research
on protein
Gene for pest
Gene used to alter
Protein dissolves
Human growth
resistance inserted bacteria for cleaning blood clots in heart hormone treats
into plants
up toxic waste
attack therapy
stunted growth
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Using Restriction Enzymes to Make Recombinant
DNA
• Bacterial restriction enzymes cut DNA
molecules at specific DNA sequences called
restriction sites
• Yields restriction fragments
• Most useful restriction enzymes give staggered
cut  “sticky ends.”
Animation: Restriction Enzymes
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Education, Inc.
• Sticky ends bond with complementary sticky ends
of other fragments
• DNA ligase seals bonds between fragments
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2011 Pearson
Education, Inc.
Figure 20.3-1
Restriction site
5
GAATTC
CTTAAG
DNA
3
5
3
1 Restriction enzyme
cuts sugar-phosphate
backbones.
5
3
3
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
5
5 Sticky 3
end
3
5
Figure 20.3-2
Restriction site
5
3
GAATTC
CTTAAG
DNA
5
3
1 Restriction enzyme
cuts sugar-phosphate
backbones.
5
5
3
3
5 Sticky 3
3
5
end
5
2 DNA fragment added
3
3
5
from another molecule
cut by same enzyme.
Base pairing occurs.
5
3 5
3 5
G AATT C
C TTAA G
G AATT C
C TTAA G
53
5 3
3
One possible combination
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3
5
Figure 20.3-3
Restriction site
5
3
GAATTC
CTTAAG
DNA
5
3
1 Restriction enzyme
cuts sugar-phosphate
backbones.
5
3
5
3
5 Sticky 3
3
5
end
5
2 DNA fragment added
3
3
5
from another molecule
cut by same enzyme.
Base pairing occurs.
5
3 5
G AATT C
C TTAA G
53
5 3
3
3 DNA ligase
3 5
G AATT C
C TTAA G
3
5
One possible combination
seals strands
5
3
3
Recombinant DNA molecule
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5
Cloning a Eukaryotic Gene in a Bacterial
Plasmid
• A cloning vector (original plasmid)  DNA
molecule that carries foreign DNA into a host cell
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Education, Inc.
Figure 20.4
TECHNIQUE
Bacterial plasmid
R
amp gene
Hummingbird cell
lacZ gene
Restriction
site
Sticky
ends
Gene of
interest
Hummingbird DNA
fragments
Recombinant plasmids Nonrecombinant
plasmid
Bacteria carrying
plasmids
RESULTS
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying
recombinant
plasmid
with disrupted
lacZ gene
One of many
bacterial
clones
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Figure 20.4a-1
TECHNIQUE
Bacterial plasmid
ampR gene
Hummingbird cell
lacZ gene
Restriction
site
Sticky
ends
Gene of
interest
Hummingbird DNA
fragments
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Figure 20.4a-2
TECHNIQUE
Bacterial plasmid
ampR gene
Hummingbird cell
lacZ gene
Restriction
site
Sticky
ends
Gene of
interest
Hummingbird DNA
fragments
Recombinant plasmids Nonrecombinant
plasmid
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Figure 20.4a-3
TECHNIQUE
Bacterial plasmid
ampR gene
Hummingbird cell
lacZ gene
Restriction
site
Sticky
ends
Gene of
interest
Hummingbird DNA
fragments
Recombinant plasmids Nonrecombinant
plasmid
Bacteria carrying
plasmids
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Figure 20.4b
Bacteria carrying
plasmids
RESULTS
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying
recombinant
plasmid
with disrupted
lacZ gene
One of many
bacterial
clones
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Storing Cloned Genes in DNA Libraries
• A genomic library is made using plasmids or
bacteriophages
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Education, Inc.
Figure 20.5
Foreign genome
Cut with restriction enzymes into either
small
large
or
Bacterial artificial
fragments
fragments
chromosome (BAC)
Large
insert
with
many
genes
Recombinant
plasmids
(b) BAC clone
Plasmid
clone
(a) Plasmid library
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(c) Storing genome libraries
Complementary DNA (cDNA) library
• Made by cloning DNA made in vitro by reverse
transcription of all the mRNA produced by a
particular cell
• A cDNA library represents only the subset of
genes transcribed into mRNA in the original cells
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Education, Inc.
Figure 20.6-1
DNA in
nucleus
mRNAs in
cytoplasm
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Figure 20.6-2
DNA in
nucleus
mRNAs in
cytoplasm
Reverse
transcriptase Poly-A tail
mRNA
A A A A A A 3
5
3
T T T T T 5
DNA Primer
strand
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Figure 20.6-3
DNA in
nucleus
mRNAs in
cytoplasm
Reverse
transcriptase Poly-A tail
mRNA
A A A A A A 3
5
3
T T T T T 5
DNA Primer
strand
5
3
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A A A A A A 3
T T T T T 5
Figure 20.6-4
DNA in
nucleus
mRNAs in
cytoplasm
Reverse
transcriptase Poly-A tail
mRNA
A A A A A A 3
5
3
T T T T T 5
DNA Primer
strand
A A A A A A 3
T T T T T 5
5
3
5
3
DNA
polymerase
3
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
5
Figure 20.6-5
DNA in
nucleus
mRNAs in
cytoplasm
Reverse
transcriptase Poly-A tail
mRNA
A A A A A A 3
5
3
T T T T T 5
DNA Primer
strand
A A A A A A 3
T T T T T 5
5
3
5
3
DNA
polymerase
3
5
3
5
5
3
cDNA
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Screening a Library for Clones Carrying a Gene of
Interest
• Identified with a nucleic acid probe having a
sequence complementary to the gene
• Process is called nucleic acid hybridization
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Education, Inc.
• A probe can be synthesized that is
complementary to the gene of interest
• For example, if the desired gene is
5
 CTCATCACCGGC
3
GAGTAGTGGCCG
5
– Then we3would
synthesize this probe
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2011 Pearson
Education, Inc.
Figure 20.7
Radioactively
labeled probe
molecules
TECHNIQUE
Gene of
interest
Probe
DNA
Multiwell plates
holding library
clones
Nylon membrane
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
5
3
 CTCATCACCGGC
GAGTAGTGGCCG
5
3
Singlestranded
DNA from
cell
Location of
DNA with the
complementary
sequence
Film
Nylon
membrane
Eukaryotic Cloning and Expression Systems
• Avoid eukaryote-bacterial incompatibility issues by
using eukaryotic cells, such as yeasts or cultured
mammal cells, as hosts for cloning and expressing
genes
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• Electroporation: applying a brief electrical pulse
to create temporary holes in plasma membranes
 introduces recombinant DNA into eukaryotic
cells
• Alternatively, scientists can inject DNA into cells
using microscopically thin needles
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Cross-Species Gene Expression and Evolutionary
Ancestry
• The remarkable ability of bacteria to express some
eukaryotic proteins underscores the shared
evolutionary ancestry of living species
• e.g., Pax-6 (gene that directs formation of
vertebrate eye); also directs the formation of
insect eye
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Amplifying DNA in Vitro: The Polymerase Chain
Reaction (PCR)
• Polymerase chain reaction, PCR produce
many copies of a specific target segment of DNA
• Key to PCR is an unusual, heat-stable DNA
polymerase called Taq polymerase.
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Education, Inc.
Figure 20.8
5
TECHNIQUE
3
Target
sequence
Genomic DNA
1 Denaturation
3
5
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
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Figure 20.8a
5
TECHNIQUE
3
Target
sequence
Genomic DNA
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3
5
Figure 20.8b
1 Denaturation
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
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Figure 20.8c
Cycle 2
yields
4
molecules
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Figure 20.8d
Cycle 3
yields 8
molecules;
2 molecules
(in white boxes)
match target
sequence
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Gel Electrophoresis and Southern Blotting
• Gel electrophoresis  method of rapidly
analyzing and comparing genomes
• Uses a gel as a molecular sieve to separate
nucleic acids or proteins by size, electrical charge,
and other properties
Animation: Biotechnology Lab
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2011 Pearson
Education, Inc.
Figure 20.9
TECHNIQUE
1
Mixture of
DNA molecules of
different
sizes
Power
source
 Cathode
Anode 
Wells
Gel
2

Power
source

Longer
molecules
Shorter
molecules
RESULTS
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Figure 20.9a
TECHNIQUE
1
Mixture of
DNA molecules of
different
sizes
Power
source
 Cathode
Anode 
Wells
Gel
2

Power
source

Longer
molecules
Shorter
molecules
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Figure 20.9b
RESULTS
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• Restriction fragment analysis  DNA fragments
produced by restriction enzyme digestion sorted
by gel electrophoresis
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2011 Pearson
Education, Inc.
• Variations in DNA sequence are called
polymorphisms
• Sequence changes that alter restriction sites are
called RFLPs (restriction fragment length
polymorphisms)
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2011 Pearson
Education, Inc.
Figure 20.10
Normal -globin allele
175 bp
DdeI
Large fragment
201 bp
DdeI
Normal Sickle-cell
allele
allele
DdeI
DdeI
Large
fragment
Sickle-cell mutant -globin allele
376 bp
376 bp
DdeI
201 bp
175 bp
Large fragment
DdeI
DdeI
(a) DdeI restriction sites in normal and
sickle-cell alleles of the -globin gene
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(b) Electrophoresis of restriction
fragments from normal and
sickle-cell alleles
Figure 20.10a
Normal -globin allele
175 bp
DdeI
Large fragment
201 bp
DdeI
DdeI
DdeI
Sickle-cell mutant -globin allele
Large fragment
376 bp
DdeI
DdeI
DdeI
(a) DdeI restriction sites in normal and
sickle-cell alleles of the -globin gene
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Figure 20.10b
Normal Sickle-cell
allele
allele
Large
fragment
376 bp
201 bp
175 bp
(b) Electrophoresis of restriction
fragments from normal and
sickle-cell alleles
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• Southern blotting combines gel electrophoresis
of DNA fragments with nucleic acid hybridization
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Education, Inc.
Figure 20.11
TECHNIQUE
DNA  restriction enzyme
Restriction
fragments
I
II III
Heavy
weight
Nitrocellulose
membrane (blot)
Gel
Sponge
I Normal II Sickle-cell III Heterozygote
-globin allele
allele
1 Preparation of
restriction fragments
I
Alkaline
solution
2 Gel electrophoresis
II III
Radioactively labeled
probe for -globin
gene
Nitrocellulose blot
4 Hybridization with labeled probe
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Paper
towels
3 DNA transfer (blotting)
Probe base-pairs
with fragments
Fragment from
sickle-cell
-globin allele
Fragment from
normal - globin
allele
I
II III
Film
over
blot
5 Probe detection
DNA Sequencing
• Relatively short DNA fragments can be sequenced
by the dideoxy chain termination method, the first
automated method to be employed
• Modified nucleotides called
dideoxyribonucleotides (ddNTP) attach to
synthesized DNA strands of different lengths
• Each type of ddNTP is tagged with a distinct
fluorescent label that identifies the nucleotide at
the end of each DNA fragment
• The DNA sequence can be read from the resulting
spectrogram
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2011 Pearson
Education, Inc.
Figure 20.12
TECHNIQUE
DNA
(template strand)
5 C
3
5
3
T
G
A
C
T
T
C
G
A
C
A
A
Primer Deoxyribonucleotides Dideoxyribonucleotides
T 3
(fluorescently tagged)
G
T
T
5
DNA
polymerase
dATP
ddATP
dCTP
ddCTP
dTTP
ddTTP
dGTP
ddGTP
P P P
P P P
G
OH
DNA (template
C strand)
T
G
A
C
T
T
C
ddG
C
G
ddC
T
A
T
C
G
G
A
T
T
T
A
T
ddA
G
C
T
G
T
T
ddA
A
G
C
T
G
T
T
ddG
A
A
G
C
T
G
T
T
Shortest
Direction
of movement
of strands
Longest labeled strand
Detector
Laser
Shortest labeled strand
RESULTS
Last nucleotide
of longest
labeled strand
Last nucleotide
of shortest
labeled strand
H
Labeled strands
ddT
G
A
A
G
C
T
G
T
T
G
A
C
T
G
A
A
G
C
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G
ddC
T
G
A
A
G
C
T
G
T
T
ddA
C
T
G
A
A
G
C
T
G
T
T
ddG
A
C
T
G
A
A
G
C
T
G
T
T
3
5
Longest
Figure 20.12a
TECHNIQUE
DNA
(template strand)
5
3
C
T
G
A
C
T
T
C
G
A
C
A
A
Primer Deoxyribonucleotides Dideoxyribonucleotides
T 3
(fluorescently tagged)
G
T
T
5
DNA
polymerase
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dATP
ddATP
dCTP
ddCTP
dTTP
ddTTP
dGTP
ddGTP
P P P
G
OH
P P P
G
H
Figure 20.12b
TECHNIQUE (continued)
5
3
DNA (template
C strand)
T
G
A
C
T
T
C
G
A
C
A
A
ddC
T
G
T
T
ddG
C
T
G
T
T
Labeled strands
ddA
G
C
T
G
T
T
ddA
A
G
C
T
G
T
T
ddG
A
A
G
C
T
G
T
T
ddT
G
A
A
G
C
T
G
T
T
ddC
T
G
A
A
G
C
T
G
T
T
Shortest
Direction
of movement
of strands
3
5
Longest
Longest labeled strand
Detector
Laser
ddA
C
T
G
A
A
G
C
T
G
T
T
ddG
A
C
T
G
A
A
G
C
T
G
T
T
Shortest labeled strand
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Figure 20.12c
Direction
of movement
of strands
Longest labeled strand
Detector
Laser
Shortest labeled strand
RESULTS
Last nucleotide
of longest
labeled strand
Last nucleotide
of shortest
labeled strand
G
A
C
T
G
A
A
G
C
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• Reverse transcriptase-polymerase chain
reaction (RT-PCR)
• Reverse transcriptase + mRNA  cDNA, which
serves as a template for PCR amplification of the
gene of interest
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2011 Pearson
Education, Inc.
Figure 20.13
TECHNIQUE
1 cDNA synthesis
mRNAs
cDNAs
2 PCR amplification
Primers
-globin
gene
3 Gel electrophoresis
RESULTS
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Embryonic stages
1 2 3 4 5 6
Expression of Interacting Groups of Genes
• DNA microarray assays  compare patterns of
gene expression in different tissues, at different
times, or under different conditions
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2011 Pearson
Education, Inc.
Figure 20.15
TECHNIQUE
1 Isolate mRNA.
2 Make cDNA by reverse
transcription, using
fluorescently labeled
nucleotides.
3 Apply the cDNA mixture to a
microarray, a different gene
in each spot. The cDNA hybridizes
with any complementary DNA on
the microarray.
Tissue sample
mRNA molecules
Labeled cDNA molecules
(single strands)
DNA fragments
representing a
specific gene
DNA microarray
4 Rinse off excess cDNA; scan microarray
for fluorescence. Each fluorescent spot
(yellow) represents a gene expressed
in the tissue sample.
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DNA microarray
with 2,400
human genes
Figure 20.15a
DNA microarray
with 2,400
human genes
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Determining Gene Function
• in vitro mutagenesis  mutations are introduced
into a cloned gene, altering or destroying its
function
• Mutated gene returned to the cell normal gene
function determined by examining the mutant’s
phenotype
• Gene expression can also be silenced using RNA
interference (RNAi)
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• Genetic markers called SNPs (single nucleotide
polymorphisms) occur on average every 100–
300 base pairs
• SNPs can be detected by PCR, and any SNP
shared by people affected with a disorder but not
among unaffected people may pinpoint the
location of the disease-causing gene
© 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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2011 Pearson
Education, Inc.
Figure 20.16
DNA
T
Normal allele
SNP
C
Disease-causing
allele
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Cloning
• Organismal cloning produces one or more
organisms genetically identical to the “parent” that
donated the single cell
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2011 Pearson
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Cloning Plants: Single-Cell Cultures
• Totipotent cell can generate a complete new
organism
• Plant cloning is used extensively in agriculture
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2011 Pearson
Education, Inc.
Figure 20.17
Cross
section of
carrot root
2-mg
fragments
Fragments were
cultured in nutrient medium;
stirring caused
single cells to
shear off into
the liquid.
Single cells
free in
suspension
began to
divide.
Embryonic
plant developed
from a cultured
single cell.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Plantlet was
cultured on
agar medium.
Later it was
planted in soil.
Adult
plant
Cloning Animals: Nuclear Transplantation
• Nucleus of an unfertilized egg cell is replaced with
the nucleus of a differentiated cell
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2011 Pearson
Education, Inc.
Figure 20.18
EXPERIMENT Frog embryo
Frog egg cell
Frog tadpole
UV
Less differentiated cell
Fully differentiated
(intestinal) cell
Donor
nucleus
transplanted
Donor
nucleus
transplanted
Enucleated
egg cell
Egg with donor nucleus
activated to begin
development
RESULTS
Most develop
into tadpoles.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Most stop developing
before tadpole stage.
Reproductive Cloning of Mammals
• 1997, Scottish researchers announced the birth of
Dolly, a lamb cloned from an adult sheep by
nuclear transplantation from a differentiated
mammary cell
© 2005 Pearson Education, Inc. publishing as Benjamin Cummings
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2011 Pearson
Education, Inc.
Figure 20.19
TECHNIQUE
Mammary
cell donor
Egg cell
donor
1
Cultured
mammary
cells
2
Egg
cell from
ovary
3 Cells fused
4 Grown in culture
Nucleus
removed
Nucleus from
mammary cell
Early embryo
5 Implanted in uterus
of a third sheep
Surrogate
mother
6 Embryonic
development
RESULTS
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Lamb (“Dolly”) genetically
identical to mammary cell donor
Figure 20.19a
TECHNIQUE
Mammary
cell donor
Egg cell
donor
1
Egg
cell from
ovary
Cultured
mammary
cells
2
Nucleus
removed
3 Cells fused
Nucleus from
mammary cell
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Figure 20.19b
Nucleus from
mammary cell
4 Grown in culture
Early embryo
5 Implanted in uterus
of a third sheep
Surrogate
mother
6 Embryonic
development
RESULTS
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Lamb (“Dolly”) genetically
identical to mammary cell donor
• Since 1997, cloning has been demonstrated in
many mammals, including mice, cats, cows,
horses, mules, pigs, and dogs
• CC (for Carbon Copy) was the first cat cloned;
however, CC differed somewhat from her female
“parent”
• Cloned animals do not always look or behave
exactly the same
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Figure 20.20
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Stem Cells of Animals
• Relatively unspecialized cell that can reproduce
itself indefinitely and differentiate into specialized
cells of one or more types
• Stem cells isolated from early embryos at the
blastocyst stage are called embryonic stem (ES)
cells; these are able to differentiate into all cell
types
• The adult body also has stem cells, which replace
nonreproducing specialized cells
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Figure 20.21
Embryonic
stem cells
Adult
stem cells
Cells generating
some cell types
Cells generating
all embryonic
cell types
Cultured
stem cells
Different
culture
conditions
Different
types of
differentiated
cells
Liver
cells
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Nerve
cells
Blood
cells
Practical applications of DNA technology
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Medical Applications
• Identification of human genes in which mutation
plays a role in genetic diseases
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Diagnosis and Treatment of Diseases
• Can diagnose PCR and sequence-specific
primers, then sequencing the amplified product to
look for the disease-causing mutation
• SNPs may be associated with a disease-causing
mutation
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Human Gene Therapy
• The alteration of an afflicted individual’s genes
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Figure 20.23
Cloned gene
1 Insert RNA version of normal allele
into retrovirus.
Viral RNA
Retrovirus
capsid
2 Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
3 Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
4 Inject engineered
cells into patient.
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Bone
marrow
Pharmaceutical Products
• Advances in DNA technology and genetic
research are important to the development of new
drugs to treat diseases
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Protein Production by “Pharm” Animals
• Transgenic animals are made by introducing
genes from one species into the genome of
another animal
 pharmaceutical “factories”
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Figure 20.24
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Forensic Evidence and Genetic Profiles
• An individual’s unique DNA sequence, or genetic
profile, can be obtained by analysis of tissue or
body fluids
• Can use PCR and/ or Southern Blotting/ RFLP
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Figure 20.25
(a) This photo shows
Washington just before
his release in 2001,
after 17 years in prison.
Source of
sample
STR
marker 1
STR
marker 2
STR
marker 3
Semen on victim
17,19
13,16
12,12
Earl Washington
16,18
14,15
11,12
Kenneth Tinsley
17,19
13,16
12,12
(b) These and other STR data exonerated Washington
and led Tinsley to plead guilty to the murder.
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Environmental Cleanup
• Modified microorganisms can be used to extract
minerals from the environment or degrade
potentially toxic waste materials
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Agricultural Applications
• DNA technology is being used to improve
agricultural productivity and food quality
• Genetic engineering of transgenic animals speeds
up the selective breeding process
• Beneficial genes can be transferred between
varieties of species
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• The Ti plasmid is the most commonly used vector
for introducing new genes into plant cells
• Genetic engineering in plants has been used to
transfer many useful genes including those for
herbicide resistance, increased resistance to
pests, increased resistance to salinity, and
improved nutritional value of crops
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Figure 20.26
TECHNIQUE
Agrobacterium tumefaciens
Ti
plasmid
Site where
restriction
enzyme cuts
T DNA
DNA with
the gene
of interest
RESULTS
Recombinant
Ti plasmid
Plant with new trait
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Safety and Ethical Questions Raised by DNA
Technology
• Potential benefits of genetic engineering must
be weighed against potential hazards of
creating harmful products or procedures
• Guidelines are in place in the United States
and other countries to ensure safe practices for
recombinant DNA technology
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• Genetically modified (GM) organisms
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