Download 2/24/12 Genetic Engineering

11.1 Restriction and Modification Enzymes
• Genetic engineering: using in vitro techniques
to alter genetic material in the laboratory
– Basic techniques include
• Restriction enzymes
•
•
•
•
•
Gel electrophoresis
Nucleic acid hybridization
Nucleic acid probes
Molecular cloning
Cloning vectors
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11.1 Restriction and Modification Enzymes
• Restriction enzymes: recognize specific DNA
sequences and cut DNA at those sites
– Widespread among prokaryotes
– Rare in eukaryotes
– Protect prokaryotes from hostile foreign DNA
(e.g., viral genomes)
– Essential for in vitro DNA manipulation
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11.1 Restriction and Modification Enzymes
• Three classes of restriction enzymes
– Type II cleave DNA within their recognition
sequence and are most useful for specific DNA
manipulation (Figure 11.1a)
• Restriction enzymes recognize inverted repeat
sequences (palindromes)
– Typically 4–8 base pairs long; EcoRI recognizes a
6-base-pair sequence
• Sticky ends or blunt ends
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Figure 11.1a
Single-stranded
“sticky” ends
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11.1 Restriction and Modification Enzymes
• Restriction enzymes protect cell from invasion
from foreign DNA
– Destroy foreign DNA
– Must protect their own DNA from inadvertent
destruction
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11.1 Restriction and Modification Enzymes
• Modification enzymes: protect cell’s DNA
for restriction enzymes
– Chemically modify nucleotides in restriction
recognition sequence
– Modification generally consists of methylation
of DNA (Figure 11.1b)
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Figure 11.1b
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11.1 Restriction and Modification Enzymes
• Gel electrophoresis: separates DNA molecules
based on size (Figure 11.2a)
– Electrophoresis uses an electrical field to separate
charged molecules
– Gels are usually made of agarose, a
polysaccharide
– Nucleic acids migrate through gel toward the
positive electrode due to their negatively charged
phosphate groups
• Gels can be stained with ethidium bromide
and DNA can be visualized under UV light
(Figure 11.2b)
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Figure 11.2a
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
Figure 11.2b
A
Size in base
pairs
5000
4000
B
C
D
Size in base
pairs
3000
2000
1800
1000
500

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11.1 Restriction and Modification Enzymes
• The same DNA that has been cut with
different restriction enzymes will have
different banding patterns on an agarose gel
• Size of fragments can be determined by
comparison to a standard
• Restriction map: a map of the location of
restriction enzyme cuts on a segment of DNA
(Figure 11.3)
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11.2 Nucleic Acid Hybridization
• Nucleic acid hybridization: base pairing of single
strands of DNA or RNA from two different sources
to give a hybrid double helix
– Segment of single-stranded DNA that is used in
hybridization and has a predetermined identity is
called a nucleic acid probe
• Southern blot: a hybridization procedure where
DNA is in the gel and probe is RNA or DNA
– Northern blot: RNA is in the gel
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Figure 11.4
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11.3 Essentials of Molecular Cloning
• Molecular cloning: isolation and incorporation of
a piece of DNA into a vector so it can be
replicated and manipulated
• Three main steps of gene cloning (Figure 11.5):
1. Isolation and fragmentation of source DNA
2. Insertion of DNA fragment into cloning vector
3. Introduction of cloned DNA into host organism
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Figure 11.5
Foreign DNA
Cut with restriction
enzyme
Add vector cut
with same
restriction enzyme
Sticky
ends
Vector
Add DNA ligase to
form recombinant
molecules
Cloned
DNA
Introduction of recombinant
vector into a host
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11.3 Essentials of Molecular Cloning
1. Isolation and fragmentation of source DNA
– Source DNA can be genomic DNA, RNA, or
PCR-amplified fragments
• Genomic DNA must first be restriction digested
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11.3 Essentials of Molecular Cloning
2. Insertion of DNA fragment into cloning vector
– Most vectors are derived from plasmids or
viruses
– DNA is generally inserted in vitro
– DNA ligase: enzyme that joins two DNA
molecules
• Works with sticky or blunt ends
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11.3 Essentials of Molecular Cloning
3. Introduction of cloned DNA into host organism
– Transformation is often used to get recombinant
DNA into host
– Some cells will contain desired cloned gene,
while other cells will have other cloned genes
• Gene library: mixture of cells containing a variety
of genes
– Shotgun cloning: gene libraries made by cloning
random genome fragments
Animation: Recombinant DNA
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11.3 Essentials of Molecular Cloning
• Essential to detect the correct clone
• Initial screen: antibiotic resistance, plaque
formation
– Often sufficient for cloning of PCR-generated
DNA sequences
• If working with a heterogeneous gene library you
may need to look more closely
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Figure 11.6
Transformant colonies
growing on agar surface
Replica-plate onto
membrane filter
Lyse bacteria and denature
DNA; add RNA or DNA
probe (radioactive); wash
out unbound radioactivity
Partially lyse cells; add
specific antibody; add agent
to detect bound antibody in
radiolabeled form
Autoradiograph
to detect
radioactivity
X-ray film
Positive
colonies
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11.4 Molecular Methods for Mutagenesis
• Synthetic DNA
– Systems are available for de novo synthesis
of DNA
– Oligonucleotides of 100 bases can be made
– Multiple oligonucleotides can be ligated together
– Synthesized DNA is used for primers and probes,
and in site-directed mutagenesis
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11.4 Molecular Methods for Mutagenesis
• Conventional mutagens produce mutations at
random
• Site-directed mutagenesis: performed in vitro
and introduces mutations at a precise location
(Figure 11.7)
– Can be used to assess the activity of specific
amino acids in a protein
– Structural biologists have gained significant
insight using this tool
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Figure 11.7
Clone into
single-stranded
vector
Source
Single-stranded
DNA from M13
phage
Base-pairing
with source
gene
Add synthetic
oligonucleotide
with one base
mismatch
Extend single
strand with
DNA polymerase
Transformation
and selection
Clone and
select mutant
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11.4 Molecular Methods for Mutagenesis
• Cassette mutagenesis and knockout mutations
– DNA fragment can be cut, excised, and replaced
by a synthetic DNA fragment (DNA cassettes or
cartridges)
– The process is known as cassette mutagenesis
• Gene disruption is when cassettes are inserted into
the middle of the gene (Figure 11.8)
• Gene disruption causes knockout mutations
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Figure 11.8
Gene X
EcoRI cut sites ()
Kanamycin cassette
Cut with EcoRI
and ligate
BamHI
cut site
Cut with BamHI and
transform into cell
with wild-type gene X
Linearized plasmid
Sites of recombination
Chromosome
Recombination and selection
for kanamycin-resistant cells
Gene X knockout
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Figure 11.9
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Figure 11.10
Target gene
Promoter
Coding sequence
Reporter gene
Promoter
Coding sequence
Cut and ligate
Gene fusion
Promoter
Reporter is expressed under
control of target gene promoter
Reporter
enzyme
Substrate
Colored product
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11.6 Plasmids as Cloning Vectors
• Plasmids are natural vectors and have useful
properties as cloning vectors
– Small size; easy to isolate DNA
– Independent origin of replication
– Multiple copy number; get multiple copies of
cloned gene per cell
– Presence of selectable markers
• Vector transfer carried out by chemical
transformation or electroporation
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11.6 Plasmids as Cloning Vectors
• pUC19 is a common cloning vector (Figure 11.11)
– Modified ColE1 plasmid
• Contains ampicillin resistance and lacZ genes
• Contains polylinker (multiple cloning site) within
lacZ gene
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Figure 11.11
Order of restriction
enzyme cut sites in
polylinker
Ampicillin
resistance
lacZ
ApoI - EcoRI
BanII - SacI
Acc651 - KpnI
AvaI - BsoBI SmaI - XmaI
BamHI
XbaI
AccI - HincII - SalI
BspMI - BfuAI
SbfI
PstI
SphI
HindIII
Polylinker
pUC19
2686 base pairs
lacI
Origin of
DNA replication
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11.6 Plasmids as Cloning Vectors
• Blue/white screening
– Blue colonies do not have vector with foreign
DNA inserted
– White colonies have foreign DNA inserted
• Insertional inactivation: lacZ gene is inactivated
by insertion of foreign DNA (Figure 11.12)
– Inactivated lacZ cannot process Xgal; blue color
does not develop
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Figure 11.12
lacZ
AmpR
Foreign DNA
Vector
Digestion with restriction enzyme
Opened vector
Recyclized vector without insert
Join with
DNA ligase
Vector plus foreign
DNA insert
Transform into Escherichia
coli and select on ampicillin
plates containing Xgal
Transformants blue
(-galactosidase
active)
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Transformants white
(-galactosidase
inactive)
11.7 Hosts for Cloning Vectors
• Ideal hosts should be
–
–
–
–
–
Capable of rapid growth in inexpensive medium
Nonpathogenic
Capable of incorporating DNA
Genetically stable in culture
Equipped with appropriate enzymes to allow
replication of the vector
• Escherichia coli, Bacillus subtilis,
Saccharomyces cerevisiae
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Figure 11.13
Bacteria
Escherichia coli
Bacillus subtilis
Eukaryote
Saccharomyces
cerevisiae
Well-developed
genetics
Many strains
available
Best known
bacterium
Easily transformed
Nonpathogenic
Naturally secretes
proteins
Endospore formation
simplifies culture
Well-developed
genetics
Nonpathogenic
Can process mRNA
and proteins
Easy to grow
Potentially
pathogenic
Periplasm traps
proteins
Genetically unstable
Genetics less
developed than
in E. coli
Plasmids unstable
Will not replicate
most bacterial
plasmids
Advantages
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Disadvantages
11.5 Gene Fusions and Reporter Genes
• Reporter genes
– Encode proteins that are easy to detect and
assay (Figure 11.9)
• Examples: lacZ, luciferase, GFP genes
• Gene fusions
– Promoters or coding sequences of genes of
interest can be swapped with those of reporter
genes to elucidate gene regulation under various
conditions (Figure 11.10)
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11.8 Shuttle Vectors and Expression
Vectors
• Expression vectors: allow experimenter to control
the expression of cloned genes (Figure 11.16)
– Based on transcriptional control
– Allow for high levels of protein expression
– Strong promoters
• lac, trp, tac, trc, lambda PL
– Effective transcription terminators are used to
prevent expression of other genes on the plasmid
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Figure 11.16
trc promoter
lacO
S/D
lacI
Polylinker
(cloning
site)
T1
T2
Origin of
DNA replication
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Ampicillin
resistance
11.8 Shuttle Vectors and Expression
Vectors
• In T7 expression vectors, cloned genes are
placed under control of the T7 promoter
(Figure 11.17)
• Gene for T7 RNA polymerase present and under
control of easily regulated system (e.g., lac)
– T7 RNA polymerase recognizes only T7 promoters
• Transcribes only cloned genes
• Shuts down host transcription
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Figure 11.17
Induce lac
promoter with
IPTG
T7 RNA
polymerase
Gene
product
lac Gene for
operator T7 RNA
lac
polymerase
promoter
T7
promoter
Cloned
gene
pET plasmid
Chromosome
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lacl
11.8 Shuttle Vectors and Expression
Vectors
• mRNA produced must be efficiently translated
and there are problems with this always
happening
– Bacterial ribosome binding sites are not present in
eukaryotic genomes
– Differences in codon usage between organisms
– Eukaryotic genes containing introns will not be
expressed properly in prokaryotes
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