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Concept 20.1: DNA cloning yields multiple
copies of a gene or other DNA segment
• To work directly with specific genes, scientists
prepare well-defined segments of DNA in identical
copies, a process called DNA cloning
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DNA Cloning and Its Applications:
A Preview
• Most methods for cloning pieces of DNA in the
laboratory share general features, such as the use
of bacteria and their plasmids
• Plasmids are small circular DNA molecules that
replicate separately from the bacterial
chromosome
• Cloned genes are useful for making copies of a
particular gene and producing a protein product
© 2011 Pearson Education, Inc.
• Gene cloning involves using bacteria to make
multiple copies of a gene
• Foreign DNA is inserted into a plasmid, and the
recombinant plasmid is inserted into a bacterial
cell
• Reproduction in the bacterial cell results in cloning
of the plasmid including the foreign DNA
• This results in the production of multiple copies of
a single gene
© 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
Figure 20.2a
Bacterium
1 Gene inserted into
plasmid
Bacterial
Plasmid
chromosome
Recombinant
DNA (plasmid)
Recombinant
bacterium
Gene of
interest
2 Plasmid put into
bacterial cell
Cell containing
gene of interest
DNA of
chromosome
(“foreign” DNA)
Figure 20.2b
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
Using Restriction Enzymes to Make
Recombinant DNA
• Bacterial restriction enzymes cut DNA
molecules at specific DNA sequences called
restriction sites
• A restriction enzyme usually makes many cuts,
yielding restriction fragments
• The most useful restriction enzymes cut DNA in a
staggered way, producing fragments with “sticky
ends.”
Animation: Restriction Enzymes
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• Sticky ends can bond with complementary sticky
ends of other fragments
• DNA ligase is an enzyme that seals the bonds
between restriction fragments
© 2011 Pearson Education, Inc.
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
3
3 DNA ligase
3 5
G AATT C
C TTAA G
G AATT C
C TTAA G
53
5 3
3
5
One possible combination
seals strands
5
3
3
Recombinant DNA molecule
5
Cloning a Eukaryotic Gene in a
Bacterial Plasmid
• In gene cloning, the original plasmid is called a
cloning vector
• A cloning vector is a DNA molecule that can carry
foreign DNA into a host cell and replicate there
© 2011 Pearson 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
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
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
Screening a Library for Clones Carrying a
Gene of Interest
• A clone carrying the gene of interest can be
identified with a nucleic acid probe having a
sequence complementary to the gene
• This process is called nucleic acid hybridization
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• A probe can be synthesized that is
complementary to the gene of interest
• For example, if the desired gene is
5
 CTCATCACCGGC
3
– Then we would synthesize this probe
3 GAGTAGTGGCCG
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5
• The DNA probe can be used to screen a large
number of clones simultaneously for the gene of
interest
• Once identified, the clone carrying the gene of
interest can be cultured
© 2011 Pearson Education, Inc.
Problems with expressing Eukaryotic genes
in Bacteria
• Eventhough bacteria are capable of
transcribing and translating eukaryotic
genes, they do not have the capacity to
splice exons and get rid of introns. So in
order for them to be able to make a
protein, an in intronless gene has to be
inserted into bacteria
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
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
Cut
y
DNA from chromosomes
1
2
Longer
fragments
Shorter
fragments
Figure 12.11B
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
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
8
Figure 12.12
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