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 B C D
Size in base Size in base
pairs pairs
5000
4000
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

Sticky with same
ends restriction enzyme

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

Partially lyse cells; add Lyse bacteria and denature

specific antibody; add agent DNA; add RNA or DNA
to detect bound antibody in probe (radioactive); wash
radiolabeled form out unbound radioactivity

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
Source vector

Single-stranded
DNA from M13
phage

Base-pairing Add synthetic

with source oligonucleotide
gene 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
Ampicillin polylinker
resistance lacZ ApoI - EcoRI
BanII - SacI
Acc651 - KpnI
AvaI - BsoBI -
SmaI - XmaI
BamHI
Polylinker XbaI
AccI - HincII - SalI
BspMI - BfuAI
pUC19 SbfI
2686 base pairs PstI
SphI
HindIII
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

Join with
DNA ligase
Opened vector

Recyclized vector without insert Vector plus foreign

DNA insert
Transform into Escherichia
coli and select on ampicillin
plates containing Xgal

Transformants blue Transformants white

(-galactosidase (-galactosidase
active) inactive)

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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 Eukaryote
Escherichia coli Bacillus subtilis Saccharomyces
cerevisiae

Well-developed Easily transformed Well-developed

genetics Nonpathogenic genetics
Many strains Naturally secretes Nonpathogenic
available proteins Can process mRNA
Best known Endospore formation and proteins
bacterium simplifies culture Easy to grow

Potentially Genetically unstable Plasmids unstable

pathogenic Genetics less Will not replicate
Periplasm traps developed than most bacterial
proteins in E. coli plasmids

Advantages Disadvantages
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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

Ampicillin
Origin of resistance
DNA replication

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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 T7 RNA Gene

promoter with polymerase product
IPTG

lac Gene for

operator T7 RNA
lac polymerase
promoter

T7 Cloned
promoter gene
pET plasmid

Chromosome lacl

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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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