Gene Cloning

What Does It Mean: “To Clone”?What Does It Mean: “To Clone”?
Clone: a collection of molecules or cells, all identical to an
original molecule or cell
 To "clone a gene" is to make many copies of it - for example, by
replicating it in a culture of bacteria.
 Cloned gene can be a normal copy of a gene (= “wild type”).
 Cloned gene can be an altered version of a gene (= “mutant”).
 Recombinant DNA technology makes manipulating genes
possible.
02/20/15
Asheesh Kumar Pandey
(pandeyasish@gmail.com)

One basic cloning technique begins with the
insertion of a foreign gene into a bacterial plasmid.
02/20/15Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Fig. 20.1 Asheesh Kumar Pandey

The objectives of Recombinant DNA
technology include:
◦ Identifying genes
◦ Isolating genes
◦ Modifying genes
◦ Re-expressing genes in other hosts or
organisms
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Genetic engineering produces proteins
that offer advantages over proteins
isolated from other biological sources.
These advantages include:
◦ High purity
◦ High specific activity
◦ Steady supply
◦ Batch-to-batch consistency
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Case Study: The Use of RecombinantCase Study: The Use of Recombinant
DNA to Produce Human InsulinDNA to Produce Human Insulin
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Why synthesize human insulin?Why synthesize human insulin?
Patients’ immune systems do not
produce antibodies against human insulin
as they do with bovine or porcine insulin
Projected decline in the production of
animal-derived insulin
Need for a more reliable and sustainable
method of obtaining the product
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Why is insulin needed?Why is insulin needed?
Protein hormone produced by beta cells
of islets of Langerhans in the pancreas
Regulates blood sugar by allowing uptake
of glucose from bloodstream into body
cells
Patients with diabetes have insufficient
or impaired production of insulin
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Structure of InsulinStructure of Insulin
Two polypeptide chains; one with 21
amino acids and the second with 30
amino acids
Chains are linked via a disulfide bond
Gene encoding the insulin protein is
found on chromosome 11
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Recombinant DNA TechniqueRecombinant DNA Technique
Restriction enzymes
used to cut out
insulin gene and to
cut a bacterial
(E. coli) plasmid at
the same “sticky
ends”
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Mutant strains of E. coli used to avoid
bacteria attacking “foreign” genes
Insert insulin gene next to E. coli
B-galactosidase gene which controls
transcription
Bacterial cells replicate and make copies
of insulin gene
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Insulin protein is purified (B-galactosidase
removed)
Chains are mixed and disulfide bridges
form
Yeast cells provide a sterile growth
medium
Final product is Humulin - chemically
identical to human insulin
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Possible Complications of UsingPossible Complications of Using
Human InsulinHuman Insulin
hypoglycemia (low blood sugar) tends to
be more common than with animal
insulin
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The Role of RestrictionThe Role of Restriction
EndonucleasesEndonucleases
Restriction endonucleases, first
discovered in the late 1960s, are named
for preventing invasion by foreign DNA by
cutting it into pieces
These enzymes cut at sites within the
foreign DNA instead of chewing from the
ends
By cutting DNA at specific sites they
function as finely honed molecular knives
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Naming Restriction EndonucleasesNaming Restriction Endonucleases
Restriction endonucleases are named using the 1st
three letters of their name from the Latin name of
their source microorganism Hind III
◦ First letter is from the genus H from Haemophilus
◦ Next two letters are the 1st
two letters of the species
name in from influenzae
◦ Sometimes the strain designation is included
“d” from strain Rd
◦ If microorganism produces only 1 restriction enzyme,
end the name with Roman numeral I Hind I
◦ If more than one restriction enzyme is produced, the
others are numbered sequentially II, III, IV, etc.
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Restriction Endonuclease SpecificityRestriction Endonuclease Specificity
Restriction endonucleases
recognize a specific DNA
sequence, cutting ONLY at that
sequence
◦ These enzymes can recognize
4-bp, 6-bp, 8-bp sequences
◦ The frequency of cuts lessens
when the recognition
sequence is longer
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Restriction Enzyme TerminologyRestriction Enzyme Terminology
A 6-bp cutter will yield DNA fragments
averaging 4000-bp or 4 kilobases (4kb) in
length
Heteroschizomers recognize the same
DNA sequence but use a different cutting
site – they are also called isochizomers
These enzymes cut DNA strands
reproducibly in the same place, which is
extremely useful in gene analysis
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Use of Restriction EndonucleasesUse of Restriction Endonucleases
Many restriction endonucleases make
staggered cuts in the 2 DNA strands
◦ This leaves single-stranded overhangs, called
sticky ends that can base-pair together briefly
◦ This makes joining 2 different DNA molecules
together much easier
Staggered cuts occur when the
recognition sequence usually displays
twofold symmetry, palindromes
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Restriction-Modification SystemRestriction-Modification System
What prevents these enzymes
from cutting up the host
DNA?
◦ They are paired with methylases
◦ Theses enzymes recognize,
methylate the same site
Together they are called a
restriction-modification
system, R-M system
Methylation protects DNA,
after replication the parental
strand is already methylated
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Basics of type II Restriction EnzymesBasics of type II Restriction Enzymes
 No ATP requirement.
 Recognition sites in double stranded DNA have a 2-fold axis
of symmetry – a “palindrome”.
 Cleavage can leave staggered or "sticky" ends or can produce
"blunt” ends.
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Type II restriction enzymeType II restriction enzyme
nomenclaturenomenclature
 EcoRI – Escherichia coli strain R, 1st
enzyme
 BamHI – Bacillus amyloliquefaciens strain H, 1st
enzyme
 DpnI – Diplococcus pneumoniae, 1st
enzyme
 HindIII – Haemophilus influenzae, strain D, 3rd
enzyme
 BglII – Bacillus globigii, 2nd
enzyme
 PstI – Providencia stuartii 164, 1st
enzyme
 Sau3AI – Staphylococcus aureus strain 3A, 1st
enzyme
 KpnI – Klebsiella pneumoniae, 1st
enzyme
02/20/15
Why the funny names?

An Experiment Using RestrictionAn Experiment Using Restriction
EndonucleaseEndonuclease
An early experiment used EcoRI to
cut 2 plasmids, small circular pieces
of DNA independent of the host
chromosome
Each plasmid had 1 site for EcoRI
◦ Cutting converted circular plasmids
into linear DNA with the same
sticky ends
◦ The ends base pair
 Some ends re-close
 Others join the 2 pieces
DNA ligase joins 2 pieces with
covalent bonds
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Plasmids – vehicles for cloningPlasmids – vehicles for cloning
 Plasmids are naturally occurring
extrachromosomal DNA molecules.
 Plasmids are circular, double-stranded
DNA.
 Plasmids are the means by which
antibiotic resistance is often
transferred from one bacteria to
another.
 Plasmids can be cleaved by restriction
enzymes, leaving sticky or blunt ends.
 Artificial plasmids can be constructed
by linking new DNA fragments to the
sticky ends of plasmid.
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Tetr
Ampr
Ori
pBR322
4361bp
Ori
pUC18
Ampr
MCS
LacZ

Cloning VectorsCloning Vectors
 A cloning vector is a plasmid that can be
modified to carry new genes.
 Plasmids useful as cloning vectors must
have:
 An origin of replication.
 A selectable marker (antibiotic
resistance gene, such as ampr
and
tetr
).
 Multiple cloning site (MCS) (site
where insertion of foreign DNA will
not disrupt replication or inactivate
essential markers).
 Easy to purify away from host DNA.
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Tetr
Ampr
Ori
pBR322
4361bp
Ori
pUC18
Ampr
MCS
LacZ
Older cloning vector
Newer cloning vector

Insert – Target DNA
2. Restriction Enzymes
1. PCR product
RE1
RE2
RE1
RE1
RE2
RE2
T
T
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VectorsVectors
Vectors function as DNA carriers to allow
replication of recombinant DNAs
Typical experiment uses 1 vector plus a piece
of foreign DNA
◦ Depends on the vector for its replication
◦ Foreign DNA has no origin of replication, the site
where DNA replication begins
There are 2 major classes of vectors:
◦ Plasmids
◦ Phages
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Vector requirementsVector requirements
 Selectable marker
◦ Resistance to ampicillin, kanamycin, hygromycin, herbicide, etc.
◦ Allows only recombinant clones to survive
 Multiple cloning site (mcs)
◦ many (unique) restriction sites
 For vectors that amplify in E. coli cells:
◦ Origin of replication (Ori): required for plasmid to replicate in
bacterium
◦ Control of copy number (F factor plasmid): reduces copy
number of plasmids in a given cell, lessens problems of
rearrangements (chimerism)
 For vectors that amplify in yeast cells:
◦ Autonomous replication sequence (ARS): similar to Ori
◦ CEN: centromere
◦ TEL: telomere
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Plasmids As VectorsPlasmids As Vectors
pBR plasmids were developed early but
are rarely used today
pUC series is similar to pBR
◦ 40% of the DNA, including tetracycline
resistance has been deleted
◦ Cloning sites are clustered together into one
area called the multiple cloning site (MCS)
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pBR322 PlasmidpBR322 Plasmid
pBR322 illustrates
cloning methods simply
◦ Resistance for 2 antibiotics
 Tetracycline
 Ampicillin
◦ Origin of replication
between the 2 resistance
genes
◦ Only 1 site for several
restriction enzymes
02/20/15

pBR322 CloningpBR322 Cloning
Clone a foreign DNA into the
PstI site of pBR322
 Cut the vector to generate
the sticky ends
 Cut foreign DNA with PstI
also – compatible ends
 Combine vector and
foreign DNA with DNA ligase
to seal sticky ends
 Now transform the
plasmid into E. coli
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Bacterial TransformationBacterial Transformation
Traditional method involves incubating
bacterial cells in concentrated calcium
salt solution
◦ The solution makes the cell membrane leaky,
permeable to the plasmid DNA
Newer method uses high voltage to drive
the DNA into the cells in process called
electroporation
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Transformation
Two main methods:
1. Chemical transformation – Chilling cells in the presence
of Ca2+ prepares the cell walls to become permeable
to plasmid DNA. Cells are briefly heat shocked which
causes the DNA to enter the cell
2. Electoporation- making holes in bacterial cells, by
briefly shocking them with an electric field of 10-
20kV/cm. Plasmid DNA can enter the cell through
these holes.
Use of bacterial cells to amplify
the DNA of interest
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Screening TransformantsScreening Transformants
Transformation produces bacteria with:
◦ Religated plasmid
◦ Religated insert
◦ Recombinants
Identify the recombinants using the antibiotic
resistance
◦ Grow cells with tetracycline so only cells with plasmid
grow, not foreign DNA only
◦ Next, grow copies of the original colonies with ampicillin
which kills cells with plasmid including foreign DNA
02/20/15

Screening With Replica PlatingScreening With Replica Plating
Replica plating transfers clone
copies from original
tetracycline plate to a plate
containing ampicillin
A sterile velvet transfer tool
can be used to transfer copies
of the original colonies
Desired colonies are those
that do NOT grow on the new
ampicillin plate
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pUC andpUC and ββ-galactosidase-galactosidase
Newer pUC plasmids have:
◦ Ampicillin resistance gene
◦ Multiple cloning site inserted into the gene lacZ’
coding for the enzyme β-galactosidase
 Clones with foreign DNA in the MCS disrupt the ability of
the cells to make β-galactosidase
 Plate on media with a β-galactosidase indicator (X-gal)
and clones with intact β-galactosidase enzyme will
produce blue colonies
 Colorless colonies should contain the plasmid with
foreign DNA
02/20/15

Lac Z gene
LacZ genepromotor
RNA
pol.
Gene expression dogma
DNA
LacZ mRNA
Ribosome
β-galactosidase
RNA
Protein
X-gal BLUE coloniesBLUE colonies
WHITEWHITE coloniescoloniesX-gal
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Expression vectors
To yield the product of a cloned gene for further studies
1) Expression vectors with a strong promoter
More mRNA More protein
2) Expression vectors with an inducible promoter
Foreign proteins when overexpressed could be toxic
Keep the gene expression off till it is time to turn it on
a. Drug-inducible (e.g. IPTG)
b. Heat-inducible
02/20/15

Expression vectors
To yield the product of a cloned gene for further studies
1) Expression vectors with a strong promoter
More mRNA More protein
2) Expression vectors with an inducible promoter
Foreign proteins when overexpressed could be toxic
Keep the gene expression off till it is time to turn it on
a. Drug-inducible (e.g. IPTG or arabinose)
b. Heat-inducible
3) Expression vectors with a fusion tag for affinity purification
Facilitate the purification of the expressed protein
1) 6 Histidine tag
2) Glutathione transferase tag (GST)
3) Maltose-binding protein tag
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LacZpromotor operator
Repressor
Lac Z gene
LacZpromotor
RNA
pol.
RNA
pol.
IPTG
IPTG
IPTG
LacZpromotor operator
IPTG
IPTG
RNA
pol.
IPTG
X-gal
Β-galactosidase
X + galactose
Cells which produce ß-galactosidase form BLUE coloniesBLUE colonies.
Cells without ß-galactosidase production form WHITEWHITE coloniescolonies.
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X
XX
X
X
Plasmid without Insert
Plasmid +Insert
without plasmid
Screening
LacZ
pGEM
Insert
WHITE coloniesWHITE colonies BLUEBLUE coloniescolonies
prom
otor
operat
or
T
T
02/20/15

An Expression Vector for Making a LacZ Fusion Protein
LacZ can be replaced with:
1) Glutathione S-transferase (GST)
2) Maltose-binding protein (MBP)
Affinity Ligand:
Glutathione
Starch (amylose)02/20/15

A plasmid DNA will be purified from the bacteria cells.
Insert
Vector
Confirmation by digestion with restriction
enzyme and separation of the digestion
products on agarose gel
EcoRI
EcoRI
Plasmid DNA will be digested with EcoRI, and analyzed by gel
electrophoresis for identification of the clone containing insert.
pGEM
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Directional CloningDirectional Cloning
Cut a plasmid with 2 restriction enzymes
from the MCS
Clone in a piece of foreign DNA with 1
sticky end recognizing each enzyme
The insert DNA is placed into the vector
in only 1 orientation
Vector religation is also prevented as the
two restriction sites are incompatible
02/20/15

SummarySummary
First generation plasmid cloning vectors include
pBR322 and the pUC plasmids
pBR322 has
◦ 2 antibiotic resistance genes
◦ Variety of unique restriction sites for inserting foreign DNA
◦ Most of these sites interrupt antibiotic resistance, making
screening straightforward
pUC has
◦ Ampicillin resistance gene
◦ MCS that interrupts a β-galactosidase gene
MCS facilitates directional cloning into 2 different
restriction sites
02/20/15

Relaxed, cccDNA
(covalently, closed, circular)
Supercoiled
DNA
Open (nicked) circular
Gyrase
Topoisomerase
Endonuclease
DNA ligase
Endonuclease
Interconversions of
different forms of plasmids
Relaxed Plasmid (multiple copies per cell)
Stringent Plasmid (limited copies per cell)
Non-integrative
Episome (Integrated)
tra genes (conjugative / non-conjugative)
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Phages As VectorsPhages As Vectors
Bacteriophages are natural vectors that
transduce bacterial DNA from one cell to
another
Phage vectors infect cells much more
efficiently than plasmids transform cells
Clones are not colonies of cells using
phage vectors, but rather plaques, a
clearing of the bacterial lawn due to
phage killing the bacteria in that area
02/20/15

Vectors Host - Features Insert Size
Plasmid E.coli- Introduced by transformation 1-5 kb
(electroporation or heat shock)
Phage E.coli- Virus that infects bacteria 10-15 kb
Introduced by transfection
Cosmid E.coli- Plasmid with “cos” sites for 30-45 kb
packaging into lambda phage particles.
Introduced by infection into E. coli
02/20/15
DNA molecule originating from a virus, a plasmid, or the cell of a higher
organism into which another DNA fragment of appropriate size can be
integrated without loss of the vector’s capacity for self-replication
Vectors introduce foreign DNA into host cells, where the DNA can be
reproduced in large quantities

RNase H
Pol I
RNA II primes
DNA synthesis
Replication
RNA II
(555 bp)
RNA I (108
bp)
5’
5’3’
3’
Origin
Infrequentl
y
Frequently
RNA I/ RNA II hybrid +
Rop dimer (initial
pairing of duplex)
Rop dimer
RNA II inactivated
for primer function
No Replication
Plasmid copy number (regulation of
replication of Col E1-derived plasmids
02/20/15

Partitioning and segregative stability of
plasmids
par gene
Incompatibility of plasmids
Inability of two different plasmids to
coexist in the same cell in the absence of
selection pressure (same mechanism of
replication control)
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λλ Phage VectorsPhage Vectors
First phage vectors were constructed by
Fred Blattner and colleagues
◦ Removed middle region
◦ Retained genes needed for phage replication
◦ Could replace removed phage genes with
foreign DNA
Originally named Charon phage
More general term, replacement vectors
02/20/15

Phage Vector AdvantagesPhage Vector Advantages
Phage vectors can receive larger amounts
of foreign DNA
◦ Charon 4 can accept up to 20kb of DNA
◦ Traditional plasmid vectors take much less
Phage vectors require a minimum size
foreign DNA piece (12 kb) inserted to
package into a phage particle
02/20/15

Cloning Using a Phage VectorCloning Using a Phage Vector
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Genomic LibrariesGenomic Libraries
A genomic library contains clones of all
the genes from a species genome
Restriction fragments of a genome can be
packaged into phage using about 16 – 20
kb per fragment
This fragment size will include the
entirety of most eukaryotic genes
Once a library is established, it can be
used to search for any gene of interest
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Plaque HybridizationPlaque Hybridization
Searching a genomic
library requires probe
showing which clone
contains desired gene
Ideal probe – labeled
nucleic acid with
sequence matching the
gene of interest
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CosmidsCosmids
Cosmids are designed for cloning large DNA fragments
◦ Behave as plasmid and phage
◦ Contain
 cos sites, cohesive ends of phage DNA that allow the DNA to be packaged
into a λ phage head
 Plasmid origin of replication permitting replication as plasmid in bacteria
◦ Nearly all λ genome removed so there is room for large inserts
(40-50 kb)
◦ So little phage DNA can’t replicate, but they are infectious
carrying recombinant DNA into bacterial cells
02/20/15

M13 Phage VectorsM13 Phage Vectors
Long, thin, filamentous phage M13
Contains:
◦ Gene fragment with β-galactosidase
◦ Multiple cloning site like the pUC family
Advantage
◦ This phage’s genome is single-stranded DNA
◦ Fragments cloned into it will be recovered in
single-stranded form
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M13 Cloning to Recover Single-M13 Cloning to Recover Single-
stranded DNA Productstranded DNA Product
 After infecting E. coli cells, single-
stranded phage DNA is converted
to double-stranded replicative
form
 Use the replicative form for cloning
foreign DNA into MCS
 Recombinant DNA infects host cells
resulting in single-stranded
recombinant DNA
 Phage particles, containing single-
stranded phage DNA is secreted
from transformed cells and can be
collected from media
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PhagemidsPhagemids
Phagemids are also vectors
◦ Like cosmids have aspects of
both phages and plasmids
◦ Has a MCS inserted into lacZ’
gene to screen blue staining /
white colonies
◦ Has origin of replication of
single-stranded phage f1 to
permit recovery of single-
stranded recombinant DNA
◦ MCS has 2 phage RNA
polymerase promoters, 1 on
each side of MCS
02/20/15

Bacterial Artificial Chromosomes(BACs) andBacterial Artificial Chromosomes(BACs) and
Yeast Artificial Chromosomes(YACs)Yeast Artificial Chromosomes(YACs)
 BACs can hold up to 300 kbs.
 The F factor of E.coli is capable of
handling large segments of DNA.
 Recombinant BACs are introduced
into E.coli by electroportation ( a brief
high-voltage current). Once in the
cell, the rBAC replicates like an F
factor.
 Example: pBAC108L
 Has a set of regulatory genes, OriS,
and repE which control F-factor
replication, and parA and parB which
limit the number of copies to one or
two.
 A chloramphenicol resistance gene,
and a cloning segment.
 YACs can hold up to 500 kbs.
 YACs are designed to replicate as
plasmids in bacteria when no foreign
DNA is present. Once a fragment is
inserted, YACs are transferred to cells,
they then replicate as eukaryotic
chromosomes.
 YACs contain: a yeast centromere, two
yeast telomeres, a bacterial origin of
replication, and bacterial selectable
markers.
 YAC plasmidYeast chromosome
 DNA is inserted to a unique restriction
site, and cleaves the plasmid with another
restriction endonuclease that removes a
fragment of DNA and causes the YAC to
become linear. Once in the cell, the rYAC
replicates as a chromosome, also
replicating the foreign DNA.
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PROPERTY P1 pBAC pucBAC pCYPAC YAC
Vector Size (kb) 31 6.5 7.2 19.3 11.5
Vector Copy # single single multiple multiple multiple
Insert Size (kb) 75-95 0-300 0-300 0-300 0-2000
Cloning Strategy 2 arms single digest single digest single digest double digest
BamHI/ScaI Bam or Hind Bam or Hind Bam/Sca link Bam/EcoRI
Cloning Method Packaging Electroporate Electroporate Electroporate Spheroplast
Maintenance (copy #) single single single single single
Chimeric Clones (%) 0 2 2 0 (24/24) 20-60
Positive Selection yes no no yes yes
Copy # Induction yes no no yes no
Large Fragment Cloning VectorsLarge Fragment Cloning Vectors
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Selected Protein Expression Systems
Living Hosts:
Escherichia coli
Saccharomyces cerevisiae
Picchia pastoris
Baculovirus
Mammalian tissue culture
Transgenic animals (flies, mice, barnyard animals)
Specialized Systems:
Xenopus oocytes
In vitro transcription-translation
Choosing a System:
-- yield
-- product purification
-- post-translational modifications
-- genetic manipulation of gene/cDNA
-- cost
-- experimental difficulty
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SummarySummary
Two kinds of phage are popular cloning vectors
‑ λ phage
‑ Has nonessential genes removed making room for inserts
- Cosmids accept DNA up to 50 kb
- M13 phage
- Has MCS
- Produces single-stranded recombinant DNA
Plasmids called phagemids also produce single-
stranded DNA in presence of helper phage
Engineered phage can accommodate inserts up to 20
kb, useful for building genomic libraries
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Eukaryotic Vectors and VeryEukaryotic Vectors and Very
High Capacity VectorsHigh Capacity Vectors
There are vectors designed for cloning genes
into eukaryotic cells
Other vectors are based on the Ti plasmid to
carry genes into plant cells
Yeast artificial chromosomes (YAC) and
bacterial artificial chromosomes (BAC) are
used for cloning huge pieces of DNA
02/20/15

Identifying a Specific Clone WithIdentifying a Specific Clone With
a Specific Probea Specific Probe
Probes are used to identify a desired clone
from among the thousands of irrelevant ones
Two types are widely used
◦ Polynucleotides also called oligonucleotides
◦ Antibodies
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Polynucleotide ProbesPolynucleotide Probes
Looking for a gene you want, might use
homologous gene from another organism
◦ If already cloned
◦ Hope enough sequence similarity to permit
hybridization
◦ Need to lower stringency of hybridization
conditions to tolerate some mismatches
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Control of HybridizationControl of Hybridization
StringencyStringency
Factors that promote separation of two strands in
a DNA double helix:
◦ High temperature
◦ High organic solvent concentration
◦ Low salt concentration
Adjust conditions until only perfectly matched DNA
strands form a duplex = high stringency
Lowering these conditions lowers stringency until
DNA strands with a few mismatches can hybridize
02/20/15

SummarySummary
Specific clones can be identified using
polynucleotide probes binding to the
gene itself
Knowing the amino acid sequence of the
a gene product permits design of a set of
oligonucleotides that encode part of the
amino acid sequence
Can be a very quick and accurate means
of identifying a particular clone
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cDNA CloningcDNA Cloning
cDNA is the abbreviation for
complementary DNA or copy DNA
A cDNA library is a set of clones
representing as many as possible of the
mRNAs in a given cell type at a given time
◦ Such a library can contain tens of thousands
of different clones
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Making a cDNA LibraryMaking a cDNA Library
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Reverse Transcriptase PrimerReverse Transcriptase Primer
Central to successful cloning is the
synthesis of cDNA from an mRNA
template using reverse transcriptase (RT),
RNA-dependent DNA polymerase
◦ RT cannot initiate DNA synthesis without a
primer
◦ Use the poly(A) tail at 3’ end of most
eukaryotic mRNA so that oligo(dT) may serve
as primer
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Ribonuclease HRibonuclease H
RT with oligo(dT) primer has made a
single-stranded DNA from mRNA
Need to start to remove the mRNA
Partially degrade the mRNA using
ribonuclease H (RNase H)
◦ Enzyme degrades RNA strand of an RNA-DNA
hybrid
◦ Remaining RNA fragments serve as primers
for “second strand” DNA using nick
translation
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Nick TranslationNick Translation
The nick translation process
simultaneously:
◦ Removes DNA ahead of a nick
◦ Synthesizes DNA behind nick
◦ Net result moves or translates the
nick in the 5’ to 3’ direction
Enzyme often used is E. coli
DNA polymerase I
◦ Has a 5’ to 3’ exonuclease activity
◦ Allows enzyme to degrade DNA
ahead of the nick
02/20/15

Trailing Terminal TransferaseTrailing Terminal Transferase
Don’t have the sticky ends of genomic DNA cleaved
with restriction enzymes
Blunt ends will ligate, but inefficient
Generate sticky ends using terminal
deoxynucleotidyl transferase (TdT), terminal
transferase with one dNTP
◦ If use dCTP with the enzyme
◦ dCMPs are added one at a time to 3’ ends of the cDNA
◦ Same technique adds oligo(dG) ends to vector
◦ Generate ligation product ready for transformation
02/20/15

Vector ChoiceVector Choice
Choice based on method used to detect
positive clones
Plasmid or phagemid like pUC or pBS will
be used with colony hybridization and a
labeled DNA probe
If λ phage like λgt11, cloned cDNA under
control of lac promoter for transcription
and translation of the cloned gene and
antibody screening
02/20/15

Rapid Amplification of cDNARapid Amplification of cDNA
EndsEnds
If generated cDNA is not full-length,
missing pieces can be filled in using rapid
amplification of cDNA ends (RACE)
Technique can be used to fill in either the
missing portion at the 5’-end (usual
problem)
Analogous technique can be used to fill in
a missing 3’-end
02/20/15

5’-RACE5’-RACE
Use RNA prep containing
mRNA of interest and the
partial cDNA
Anneal mRNA with the
incomplete cDNA
Reverse transcriptase will
copy rest of the mRNA
Tail the completed cDNA
with terminal transferase
using oligo(dC)
Second strand synthesis
primed with oligo(dG)
02/20/15

SummarySummary
Make cDNA library with synthesis of cDNAs one strand at
a time
◦ Use mRNAs from a cell as templates for 1st
strands, then 1st
strand as template for 2nd
◦ Reverse transcriptase generates 1st
strand
◦ DNA polymerase I generates the second strands
Give cDNAs oligonucleotide tails that base-pair with
complementary tails on a cloning vector
Use these recombinant DNAs to transform bacteria
Detect clones with:
◦ Colony hybridization using labeled probes
◦ Antibodies if gene product translated
Incomplete cDNA can be filled in with 5’- or 3’-RACE
02/20/15

Methods of Expressing ClonedMethods of Expressing Cloned
GenesGenes
Cloning a gene permits
Production of large quantities of a
particular DNA sequence for detailed
study
Large quantities of the gene’s product
can also be obtained for further use
◦ Study
◦ Commerce
02/20/15

Expression VectorsExpression Vectors
Vectors discussed so far are used to first put a
foreign DNA into a bacterium to replicate and
screen
Expression vectors are those that can yield
protein products of the cloned genes
◦ For high level expression of a cloned gene best
results often with specialized expression vectors
◦ Bacterial vectors have a strong promoter and a
ribosome binding site near ATG codon
02/20/15

Fusion ProteinsFusion Proteins
Some cloning vectors, pUC
and pBS, can work as
expression vectors using
lac promoter
If inserted DNA is in the
same reading frame as
interrupted gene, a fusion
protein results
◦ These have a partial β-
galactosidase sequence at
amino end
◦ Inserted cDNA protein
sequence at carboxyl end
02/20/15

Inducible Expression VectorsInducible Expression Vectors
Main function of expression vector is to yield the
product of a gene – usually more is better
For this reason, expression vectors have very strong
promoters
Prefer keep a cloned gene repressed until time to
express
◦ Large quantities of eukaryotic protein in bacteria are
usually toxic
◦ Can accumulate to levels that interfere with bacterial
growth
◦ Expressed protein may form insoluble aggregates, inclusion
bodies
02/20/15

Controlling theControlling the laclac PromoterPromoter
lac promoter is somewhat inducible
◦ Stays off until stimulated
◦ Actually repression is incomplete or leaky
◦ Some expression will still occur
To avoid this problem, express using a
plasmid or phagemid carrying its own lacI
repressor gene, such as pBS
02/20/15

Arabinose PromoterArabinose Promoter
The hybrid trc promoter combines
strength of the trp (tryptophan operon)
promoter with inducibility of lac
promoter
Promoter from ara operon, PBAD, allow fine
control of transcription
◦ Inducible by arabinose, a sugar
◦ Transcription rate varies with arabinose
concentration
02/20/15

Tightly Controlled PromoterTightly Controlled Promoter
Lambda (λ) phage promoter, PL, is tightly
controlled
Expression vectors with this promoter-
operator system are used in host cells
with temperature-sensitive λ repressor
gene
◦ Repressor functions are low temperatures
◦ Raise temperature to nonpermissive
temperature, the repressor doesn’t function
and cloned gene is expressed
02/20/15

cI repressor
Bacterial chromosome
Bacterial chromosome
Po
Ptrp λcI repressor
Transcription
No Transcription
Po PL ATG Gene of Interest
Vector
Vector
Tryptophan
trp repressor
No Transcription
λcI repressor
Transcription
ATG Gene of Interest
Po
Po
Ptrp
PL
+ Tryptophan
- Tryptophan
Regulation of gene
expression
02/20/15

SummarySummary
Expression vectors are designed to yield
the protein product of a cloned gene
When a lac inducer is added, cell begins
to make T7 polymerase which transcribes
the gene of interest
Many molecules of T7 polymerase are
made, so gene is turned on to a very high
level with abundant amount of protein
product made
02/20/15

Expression Vectors That ProduceExpression Vectors That Produce
Fusion ProteinsFusion Proteins
Most vectors express fusion proteins
◦ The actual natural product of the gene isn’t made
◦ Extra amino acids help in purifying the protein product
Oligohistidine expression vector has a short sequence
just upstream of MCS encoding 6 His
◦ Oligohistidine has a high affinity for divalent metal ions like
Ni2+
◦ Permits purification by nickel affinity chromatography
◦ His tag can be removed using enzyme enterokinase without
damage to the protein product
02/20/15

Oligohistidine Expression VectorOligohistidine Expression Vector
02/20/15

Fusion Proteins inFusion Proteins in λλgt11gt11
This phage contains
lac control region and
lacZ gene
Products of gene
correctly inserted will
be fusion proteins
with a β-galactosidase
leader
02/20/15

Antibody Screening WithAntibody Screening With λλgt11gt11
Lambda phages with cDNA
inserts are plated
Protein released are
blotted onto a support
Probe with antibody to
protein
Antibody bound to protein
from plaque is detected
with labeled protein A
Partial cDNAs can be
completed with RACE
02/20/15

SummarySummary
Expression vectors frequently produce fusion
proteins
◦ One part of the protein comes from coding
sequences in the vector
◦ Other part from sequences in the cloned gene
Many fusion proteins have advantage of
being simple to isolate by affinity
chromatography
Vector lgt11 produces fusion proteins that
can be detected in plaques with a specific
antiserum
02/20/15

Bacterial Expression SystemBacterial Expression System
ShortcomingsShortcomings
There are problems with expression of
eukaryotic proteins in a bacterial system
◦ Bacteria may recognize the proteins as foreign and
destroy them
◦ Posttranslational modifications are different in
bacteria
◦ Bacterial environment may not permit correct
protein folding
Very high levels of cloned eukaryotic proteins
can be expressed in useless, insoluble form
02/20/15

Eukaryotic Expression SystemsEukaryotic Expression Systems
Avoid bacterial expression problems by
expressing the protein in eukaryotic cell
Initial cloning done in E. coli using a shuttle
vector, able to replicate in both bacterial and
eukaryotic cells
Yeast is suited for this purpose
◦ Rapid growth and ease of culture
◦ Still a eukaryote with more appropriate
posttranslational modification
◦ Secretes protein in growth medium so easy
purification
02/20/15

Use of Baculovirus As ExpressionUse of Baculovirus As Expression
VectorVector
Viruses in this class have a large circular DNA
genome, 130 kb
Major viral structural protein is made in huge
amounts in infected cells
◦ Promoter for this protein, polyhedrin, is very active
◦ These vectors can produce up to 0.5 g of protein
per liter of medium
◦ Nonrecombinant viral DNA entering cells cannot
result in infectious virus as it lacks an essential
gene supplied by the vector
02/20/15

Animal Cell TransfectionAnimal Cell Transfection
Calcium phosphate
◦ Mix cells with DNA in a phosphate buffer
◦ Then solution of calcium salt added to form a
precipitate
◦ Cells take up the calcium phosphate crystals which
include some DNA
Liposomes
◦ DNA mixed with lipid to form liposomes, small vesicles
with some of the DNA inside
◦ DNA-bearing liposomes fuse with cell membrane
carrying DNA inside the cell
02/20/15

Choosing a Mammalian Cell Expression System
Advantages:
Cell biology (ie. Localization, cell cycle etc.)
Post-translational modification (mammalian)
Improving expression vectors and transfection methods
Disadvantages:
Low yield of expressed proteins
Relatively expensive (tissue culture costs)
Must use shuttle vectors
Key terms:
Transient transfection: introduction of episomal expression vector into
mammalian tissue culture cells for short term expression experiments.
Stable transfection: introduction and integration of vector into the host cell
chromosome for long-term expression studies.
02/20/15

SummarySummary
Foreign genes can be expressed in eukaryotic
cells
These eukaryotic systems have advantages
over prokaryotic ones
◦ Made in eukaryotic cells tend to fold properly and
are then soluble rather than aggregated into
insoluble inclusion bodies
◦ Posttranslational modifications are made in a
eukaryotic manner
02/20/15

Using the Ti Plasmid to TransferUsing the Ti Plasmid to Transfer
Genes to PlantsGenes to Plants
Genes can be introduced into plants with
vectors that can replicate in plant cells
Common bacterial vector promoters and
replication origins are not recognized by plant
cells
Plasmids are used containing T-DNA
◦ T-DNA is derived from a plasmid known as tumor-
inducing (Ti)
◦ Ti plasmid comes from bacteria that cause plant
tumors called crown galls
02/20/15

Ti Plasmid InfectionTi Plasmid Infection
Bacterium infects plant, transfers Ti
plasmid to host cells
T-DNA integrates into the plant DNA
causing abnormal proliferation of plant
cells
T-DNA genes direct the synthesis of
unusual organic acids, opines which can
serve as an energy source to the infecting
bacteria but are useless to the plant
02/20/15

Ti Plasmid Transfers Crown GallTi Plasmid Transfers Crown Gall
02/20/15

Use of the T-DNA PlasmidUse of the T-DNA Plasmid
02/20/15

Gene Cloning

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