Gene Cloning

Dr. Usman Ali Ashfaq

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 Outline

What is cloning?
How to do cloning?
Validation
Application
 What is Cloning?

 Generating identical copies of organisms, cells,

or replicating nucleic acid sequences from
organisms
 Dolly (the sheep) is a clone, but a natural
identical twin is not a clone.
 DNA sequence amplified by growth is a clone,
Identical DNA molecules produced in vitro (a
PCR rxn) is not a clone.
 DNA Cloning

• When DNA is
extracted from an
organism, all its genes
are obtained
• In gene (DNA)
cloning a particular
gene is copied
(cloned)

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 PCR animation

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 PCR

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

PCR Amplification

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 Cloning Vectors
• Cloning vectors are DNA molecules that are used to
"transport" cloned sequences between biological hosts
and the test tube.

Cloning vectors share four common properties:

1. Ability to promote autonomous replication.

2. Contain a genetic marker (usually dominant) for
selection.
3. Unique restriction sites to facilitate cloning of insert
DNA.
4. Minimum amount of nonessential DNA to optimize
cloning.

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

Cloning Vectors

Origin of replication
Antibiotic-resistant genes
Allow the host to grow on
selective media
Can selectively amplify this
specific vector in the host cell

Multiple cloning sites

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 Selective Markers

• Selective marker is required for

maintenance of plasmid in the cell.
• Because of the presence of the
selective marker the plasmid
becomes useful for the cell.
• Under the selective conditions, only
cells that contain plasmids with
selectable marker can survive
• Genes that confer resistance to
various antibiotics are used.
• Genes that make cells resistant to
ampicillin, neomycin, or
chloramphenicol are used

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

Origin of Replication

• Origin of replication is
a DNA segment
recognized by the
cellular DNA-replication
enzymes.
• Without replication
origin, DNA cannot be
replicated in the cell.

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 Multiple Cloning Sites

• Many cloning vectors contain a

multiple cloning site or
polylinker: a DNA segment with
several unique sites for
restriction endo- nucleases
located next to each other
• Restriction sites of the polylinker
are not present anywhere else in
the plasmid.
• Cutting plasmids with one of the
restriction enzymes that
recognize a site in the polylinker
does not disrupt any of the
essential features of the vector

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 Multiple Cloning Sites

• Gene to be cloned
can be introduced
into the cloning
vector at one of the
restriction sites
present in the
polylinker

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 Types of Cloning Vectors

• Different types of cloning vectors are

used for different types of cloning
experiments.
• The vector is chosen according to the
size and type of DNA to be cloned

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 Plasmid Vectors

• Plasmid vectors are used to

clone DNA ranging in size
from several base pairs to
several thousands of base
pairs (100bp -10kb).
• ColE1 based, pUC vehicles
commercially available
ones, eg pGEM3,
pBlueScript

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 Cosmid Vectors

• Purpose:
1. Clone large inserts of
DNA: size ~ 45 kb
• Features:
Cosmids are Plasmids
with one or two Lambda
Cos sites.
• Presence of the Cos
site permits in vitro
packaging of cosmid
DNA into Lambda
particles

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 Yeast Artificial Chromosomes

• Purpose:
• Cloning vehicles that propagate in eukaryotic
cell hosts as eukaryotic Chromosomes
• Clone very large inserts of DNA: 100 kb - 10
Mb
• Features:
YAC cloning vehicles are plasmids
Final chimeric DNA is a linear DNA molecule
with telomeric ends: Artificial Chromosome

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 Retroviral Vectors

• Retroviral vectors are used to introduce new or

altered genes into the genomes of human and animal
cells.
• Retroviruses are RNA viruses.
• The viral RNA is converted into DNA by the viral
reverse transcriptase and then is efficiently integrated
into the host genome
• Any foreign or mutated host gene introduced into the
retroviral genome will be integrated into the host
chromosome and can reside there practically
indefinitely.
• Retroviral vectors are widely used to study
oncogenes and other human genes.

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 Two Important Enzymes

 Restriction Enzymes:
cuts the DNA from any organism at specific sequences
of a few nucleotides, generating a reproducible set of
fragments.
 DNA Ligases:
insert DNA restriction fragments into replicating DNA
molecules producing recombinant DNA.

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

Mechanism

CIVIC, Madam Sticky Ends

(Cohesive Ends)

GAATTC G AATTC
GAATTC AATTC G

EcoRI
G AATTC
G AATTC
 Restriction Mapping of DNA

Restriction
enzymes
CK A B A+B M A B 10 kb

8 kb
A 2 kb

7 kb
B 3 kb

A 5 kb
+ 3 kb
B 2 kb

Juang RH (2004) BCbasics

 The Specific Cutting and Ligation of DNA
GAATTC GAATTC
CTTAAG CTTAAG

EcoRI
G AATTC G AATTC
CTTAA G CTTAA G

G G AATTC AATTC
CTTAA CTTAA G G
EcoRI sticky end EcoRI sticky end
DNA Ligase
G AATTC
CTTAA G
Juang RH (2004) BCbasics
 Cloning Process

• Gene of interest is cut

out with RE
• Host plasmid is cut with
same RE
• Gene is inserted into
plasmid and ligated with
ligase
• New plasmid inserted
into bacterium
(transform)
• Growth of plasmid in agar
plate

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad

 How?
ORI Cells that do
Amp R.
not take up
ORI Amp R. plasmids die on
ampicillin plates

Plasmid vector Enzymatically Mix E.coli cells with

+ insert DNA into

plasmid vector
Recombinant plasmids in presence of
plasmid CaCl2 Culture on nutrient
Transformed
E.coli cell
agar plates containing survives
DNA fragment ampicillin
to be cloned Bacterial
chromosome

Independent
plasmid
replication
Cell
multiplication
 Cloning Strategy

• Involves five steps:

Enzyme restriction digest of DNA sample.
Enzyme restriction digest of DNA plasmid vector.
Ligation of DNA sample products and plasmid vector.
Transformation with the ligation products.
Growth on agar plates with selection for antibiotic
resistance.

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad

Digestion of DNA Sample

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

Digestion of Vector

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad

Ligation of DNA Sample and
Plasmid Vector

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

Transformation of Ligation Product

• The process of transferring exogenous DNA

into cells is call “transformation”
• There are basically two general methods for
transforming bacteria. The first is a chemical
method utilizing CaCl2 and heat shock to
promote DNA entry into cells.
• A second method is called electroporation
based on a short pulse of electric charge to
facilitate DNA uptake.

Deptt. of Biotechnology, Mirpur University of Science and Technology (MUST), Mirpur, AJK.
Transformation of Ligation Product

Deptt. of Biotechnology, Mirpur University of Science and Technology (MUST), Mirpur, AJK.
 Screening

• The medium in this petri

dish contains the antibiotic
Kanamycin
• The bacteria on the right
contain Kanr, a plasmid
that is resistant to
Kanamycin, while the one
on the left has no
resistance
• Note the difference in
growth

Deptt. of Biotechnology, Mirpur University of Science and Technology (MUST), Mirpur, AJK.
 Propagation

• Once colonies are

identified, they are
cultured in broth to
increase numbers and
therefore the amount of
DNA
• Samples are also
prepared for storage at -
80 degrees. They can
be kept for many years
this way.

Deptt. of Biotechnology, Mirpur University of Science and Technology (MUST), Mirpur, AJK.
 validation methods
About 100 bp
Promoter Multi. cloning site U.P.
EcoRI BamHI
Ori.
SalI L.P.
PCR
Ab. Resis.
+
EcoRI EcoRI
Restriction
Inserted Gene
About 500bp-5kb Enzyme
Marker With Prod. Without Prod.
Marker With Prod. Without
Prod.
 Sequencing
Dideoxy: (Sanger) Manual
 Primer extension reactions in four separate tubes.
 Using a dideoxy nucleotide as the chain terminator.
 Each tube contains different dideoxy nucleotide (ddATP,
ddCTP, ddGTP, ddTTP).
 Radioactive dATP is also included in all the tubes so the
DNA products will be radioactive.
 The results is a series of fragments of different lengths.
 Finally, autoradiography is performed to visualize the
DNA fragments.
 Dideoxy: (Sanger) Manual
a) Primer extension reaction c) Electrophoresis of the Protein
TACTATGCCAGA ddA ddC ddG ddT
T
20-base primer Replication with ddTTP C
T
TACTATGCCAGA G
ATGAT G
25-base primer
C
A
T
A
G
T
b) Product of the four reactions A

Product of ddA Product of ddC

Template: TACTATGCCAGA Template: TACTATGCCAGA
rxn (21) A rxn (27) ATGATAC
(24) ATGA (31) ATGATACGGTC
(26) ATGATA

Product of ddG Product of ddA

Template: TACTATGCCAGA
Template:
rxn (23)
TACTATGCCAGA
ATG
rxn (22) AT
(25) ATGAT
(28) ATGATACG
(29) ATGATACGG (30) ATGATACGGT
(32) ATGATACGGTCT
 Chain Termination

Separates
fragments
based on size
 Sequencing

Dideoxy: (Sanger) automated

 The “manual” sequencing technique is powerful but slow,
thus Rapid automated sequencing methods are required.
 Still based on the procedure using dideoxy nucleotides,
but tagged with a different fluorescent molecule, so the
product from each tube will emit a different color
fluorescence when excited by light
 After extension reaction and chain termination, all 4
solutions are mixed and electrophoresed together in the
same lane on gel analyzed by laser beam
 The color of the fluorescent light emitted from each
oligonucleotide is detected electronically
Automated Chain Termination
Method

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

Automated Chain Termination
Method

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 Applications of cloning?

• A particular gene can be isolated and its nucleotide

sequence determined
• Control sequences of DNA can be identified &
analyzed
• Protein/enzyme/RNA function can be investigated
• Mutations can be identified, e.g. gene defects
related to specific diseases
• Organisms can be ‘engineered’ for specific
purposes, e.g. insulin production, insect resistance,
etc.

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.

 Adapted from Milstein (1980) Scientific American, Oct. p.58

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How animals are cloned?
Why Cloning of Gene?

Deptt. of Bioinformatics and Biotechnology, GC University Faislabad.