SHREE SIDDAGANGA COLLEGE OF

PHARMACY TUMKUR, KARNATAKA

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 Content
s
 History
 Definition
 Steps in cloning
o Generating DNA fragments
o Insertion of target DNA in to vector
o Introduction of recombinant DNA in to host cell

 Applications of cloning
 References

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History:
 1869 - Miescher – isolated DNA from
WBC.
 1944 - Avery - DNA is primary genetic
material.
 1953 - Watson Crick - DNA double helix
structure.
 1962 - Arber - evidence of DNA restriction
nuclease.
 1966 - Nirenberg Ochoa and Khorana -
elucidation of genetic code.
 1967 - Gellert - DNA ligase enzyme.
 1972 - 73 - DNA Cloning
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techniques.
Definition:
– Cutting a piece of DNA from one
organism and inserting it into a vector
where it can be replicated by a host
organism. (Sometimes called subcloning,
because only part of the organism’s DNA
is being cloned.)

– Using nuclear DNA from one organism to

create a second organism with the same
nuclear DNA

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 Main steps in
cloning:
•Generating DNA fragments

•Insertion of target DNA in to vector

•Introduction of recombinant DNA in to

host cell

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 Generating DNA fragments
:
 Mechanical shearing:
• High speed mixing in blender or sonicator.
• Random fragment of source DNA.
• Does not produce 5’phasphate and 3’OH
ends.
• Requires repair the ends (S1nuclease or
DNA polymerase).
• Not reproducible in nature.

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 Restriction Endonuclease Digestion:
Restriction endonuclease are the enzymes
isolated from bacteria that cut DNA at specific
recognition nucleotide sequences known as
restriction sites.
• More than 200 restriction enzymes.
• Size of DNA fragments depends upon
restriction sites present in DNA.
• It produces either sticky end or blunt ends.
• Generally same enzyme is used for the vector
and the DNA of interest.
• Reproducibility of fragment is possible.

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- Restriction enzymes can generate both sticky
ends and blunt ends after they cut the
recognition sequence

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 Reverse Transcriptase Method:
• Gene organisation of eukaryotes and
prokaryotes is different.
• Bacteria or yeast do not have necessary
splicing mechanism for removal of introns.
• Mature mRNA molecules from animal cell
can be directly transcribed in to DNA using
an enzyme reverse transcriptase.
• cDNA produced for particular protein can be
joined to vector and cloned in to host cell.

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 Hybridization Method:

• Principle: mRNA forms a complex with

complementary DNA segments from which it
has been transcribed.

• Total DNA from donor organism is isolated &

dsDNA is converted to ssDNA by
denaturation.

• Mixed with mRAN trancribed by the gene

this forms DNA-RNA complex (Hybrid).
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Cont…
• DNA-RAN complex is isolated & DNA
is separated from RNA.

• ssDNA obtained can be converted to

dsDNA by DNA polymerase I.

• Suitable for isolating genes which

exist in multiple copies.

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 Chemical Synthesis:
• The technique is termed as reverse genetics.

• Possible only when amino acid sequence of protein

of interest is known.

• For which sequence of nucleotides in mRNA &

subsequently in DNA can be determined.

• This method of generating DNA fragments for

cloning will be batter for creating novel proteins.

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IDENTIFICATION OF CLONE

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 Insertion of Target DNA into
Vector:
 Vectors:are carrier DNAs into which
foreign DNAs are spliced to make a
recombinant DNA.
Types of vectors:
• Plasmids
• Bacteriophages
• Cosmids
• BACs & YACs
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Selection of Vector:

VECTOR INSERT SIZE (kbp)

• Plasmid ~ 15.0 kbp
• λ Phage ~ 50.0 kbp
• Cosmid > 50.0 <100.0 kbp
• Bacterial artificial ~ 300.0 kbp
Chromosome (BAC)
• Yeast artificial ~ 2000 kbp
Chromosome (YAC)

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Plasmids
Plasmids are double-stranded, circular,
self-replicating, extra-chromosomal
DNA molecules.

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Cont…
• Plasmids are circular pieces of DNA
found naturally in bacteria.
• Plasmids can carry antibiotic
resistance genes, genes for
receptors, toxins or other proteins.
• Plasmids replicate separately from
the genome of the organism.
• Plasmids can be engineered to be
useful cloning vectors.
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 Isolation of bacterial plasmids
o Treat the prokaryotic cells with detergent.
o Treat the lysate with potassium acetate/ acetic
acid solution and centrifuge.
o Treat the lysate with RNAase consequently.
o Mix the lysate with phenol. Phenol & water is
separated in two layers . The water based layer
contains plasmid DNA.
o Precipitate water based layer with alcohol and
collect the plasmid DNA.

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Cont…..

Ex. Of plasmids
• pBR 322- derived from E.coli prepared
by Bolivar and Rodriguez.
• pAT 153 and pXf 3 are two derivatives
of pBR 322 having smaller size &
higher copy.
• pUC derived from E.coli initial
prepared in University of California.

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Cont…..
 Advantages of plasmids:
– Small, easy to handle
– Useful for cloning small DNA
fragments
(< 15kbp)
 Disadvantages of plasmids:
– Less useful for cloning large DNA
fragments
(> 15kbp)

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Bacteriophage
s
• Phases has linear DNA, single
break create two fragments.
• Foreign DNA can be inserted
between them & two fragments
can be joined.
• Bacteriophages act as
hypodermic syringe like and insert
DNA in to the host and replicates.
• Lytic Cycle: rapid infection
resulting in lysis of cell and
release of multiple
bacteriophages.
•Lysogenic phages: For
bacteriophage λ, DNA integrates
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Cosmids
• Cosmids posses properties of both plasmids and
bacteriophages.

• Contain a cos site of λ phage (essential for packaging

of nucleic acid into protein coat)

• plus essential features of plasmids ( plasmid origin

of replication, a gene for resistance)

• Cosmids can be constructed by adding a fragment of

phage λ DNA including cos site to plasmid.

• A foregin DNA can be inserted into cosmid DNA

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C
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 BACs: Bacterial Artificial
Chromosomes
 The construction
BAC F
plasmid, which is
large than other
plasmid used as
cloning vector.

 BACs can accept

DNA inserts of
around 300kb

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YACs:Yeast Artificial Chromosomes
• YAC are the most
sophisticated yeast
vectors.

• They have centromeric &

telomeric region of
chromosome.

• very large piece of DNA

can be cloned (particular
human DNA) ~ 2000
kbp.
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Ligation
• Ligation is the process of joining two pieces of
DNA from different sources together through
the formation of a covalent bond.

• DNA ligase is the enzyme used to catalyze

this reaction.

• These enzymes are originally isolated from

viruses, also occur in E.coli & eukaryotic cell.

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 DNA Ligases
Type Energy source Used for:
E. coli NAD (turned Sticky ends
ligase into AMP and
NMN)
T7 ligase ATP (turned Sticky ends
into AMP and
PPi)
T4 ligase ATP (turned Both sticky
into AMP and and
PPi)
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blunt ends
PRECAUTION

• The most important precaution prior to setting

up a ligation reaction is to ensure that the cut
vector is prevented from recircularization.

• This is achieved by digesting the plasmid by

the same restriction enzyme(s).

• Treatment with alkaline phosphatase which

removes the terminal phosphate groups from
the cut ends of the plasmid. This ensures that
the cut plasmid does not recircularize.

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 BLUNT END LIGATION

The blunt end DNA can be joined by :

 Homopolymer tailing

 Use of Linkers Molecule

 Use of Adaptors Molecule

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 Homopolymer
tailing
• The complementary DNA strands can be
joined together by annealing.

• Addition of oligo (dA) to 3’ ends of some

DNA molecule and addition of oligo (dT) to
3’ ends of other molecule.

• The homopolymer extension can be

synthesized by using terminal
deoxynucle- otidyltrasferase (calf
thymus).
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 Homopolymer tailing
Insert Vector
5’ 3’ 3’
5’
3’ 5’ 3’ 5’

Terminal
dATP
deoxynucleotidyltransferas dTTP
e

5’ AAA 3’ 5’ TTT 3’
3’AA 5’ 3’ TTT 5’
A

LIGATION
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 Use of Linkers Molecule
• Linkers are chemically synthesized, short,
double strand DNA molecules.

• possess restriction enzyme cleavage sites.

• they can be ligated to blunt ends of any DNA.

• Cut with specific restriction enzymes to

produce sticky end DNA fragments

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Blunt- ended insert Linker (e.g. Bam HI)
5’ 3’ 5’ P GGGATCCC OH 3’
3’ 5’ 3’ OH CCCTAGGG P 5’

Ligate to linker

GGGATCCCGGGATCCC CCCTAGGGCCCTAGG
G
CCCTAGGGCCCTAG GGGATCCCGGGATC
GG CC
Cut with Bam HI

GATCCC GG
GG CCCTAG

Ligate to vector
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cut with Bam HI
 Use of Adaptors Molecule
• Adaptors are chemically synthesized, short,
double-stranded DNA molecules.

• Adaptor contains preformed sticky or

cohesive ends.

• They are useful to be ligated to DNA

fragment with blunt ends.

• The DNA fragments held to adaptors are

finally ligated to vector DNA molecules.

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 GGGATCCCGGGATCCC
5’ 3’ CCCTAGGG
3’ 5
+ CCCTAGGG
GGGATCCCGGGATCCC
DNA Fragment ’ Adaptors

DNA ligase+ATP

GGGATCCCGGGATC CCCTAGGG
CC
CCCTAGG GGGATCCCGGGATCCC
G

Ligate to vector
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Introduction of recombinant DNA in to host
cell
• After creating new vector construct it needs to
insert into a host cell so that it can be replicate.

• Hosts are the living systems or cells in which

vector can be propagated

• The process of introducing the foreign DNA

into the host cell is called transformation.

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Cont…

• Host cell may be prokaryotic (bacteria) or

eukaryotic (fungi, animal, plant).

• Micro organisms are preferred as host

cells, since they multiply faster.
PROKARYOTIC HOST (Bacteria):
• Not every bacterial cell is able to take up
plasmid DNA.

• Bacterial cells that can take up DNA from

the environment are said to be competent.

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 Escherichia
coli:
• E.coli was the first organism used in the
DNA technology experiments.
• Is common Gram – ve bacterium of
human and animal intestine.
• Under suitable environment E.coli
multiply two times in every 20 min.
Limitations of E.coli:
- Formation of endotoxins that are toxic.

- Cannot perform post- translational

modification
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 Eukaryotic
hosts:
• Eukaryotic organisms are preferred to produce
human proteins.
• Most commonly used organism is yeast,
Saccharomyces.
• It is non pathogenic organism rutinely used in
baking industry.
Mammalian cells:
- Certain complex protein produced by
mammalian cells Eg. Tissue plasminogen
activator.
- mammalian cell possess post translation
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Some Examples of Host cells
Group Examples
Prokaryotic
Bacteria E.coli
Bacillus Subtilis
Streptomyces sp
Eukaryotic
Fungi Saccharomyces cervisiae
Aspergillus nidulans
Animals Insect cells
mammalian cells
Whole organisms
Plants Protoplasts
Intact cell
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 Method of gene
transfer
Introduction of foreign DNA in to host cell is
important in gene cloning, commonaly
employed methods are:
o Transformation
o Lipofection
o Transduction
o Electroporation

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Transformionat

• Is a method of introducing foreign

DNA in to bacterial cells (E.coli)
• Carried out in ice-cold CaCl2 (0-5c) &
subsequent heat shock (37- 45c for
about 90 sec.)
• Some times calcium phosphate or
diethyl aminoethyl dextran (DEAE-
dextran) is replaced to CaCl2 .
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 Lipofection
The liposome mediated gene transfer, is
referred as lipofection.
• It is vary efficient technique & is used for -
bacteria, animal & plant host cell.
• On treatment of DNA fragment with liposome,
the DNA get encapsulated inside liposome.
• Liposome can adhere to cell membrane &
transfer DNA fragments.
• DNA enters the cell & then to the nucleus.

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Transduction
 The foreign DNA can be packed
inside animal viruses.
 These viruses can naturally infect the
cells & introduce the DNA into host
cell.
 The transfer of DNA by this approach
is referred as transduction.

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 Electroporation
 Electroporation
is technique involves electric field
mediated membrane permeabilization.

 Itis based on the principle that high voltage pulses can

induce cell plasma membrane fuse.

 Electricshock induce cellular uptake of exogenous DNA

from the suspending solution.

 Itis simple & rapid technique for introducing genes in to

the cells of various organisms.

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 WHY CLONE A
GENE?
 A particular gene can be isolated and its
nucleotide sequence can be determined
 Coding regions and control elements like
promoters can be identified and analyzed
 Protein/enzyme/RNA function can be
investigated

 Mutations can be identified, e.g. gene

defects related to specific diseases

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Cont….

 Organisms can be ‘engineered’ for

specific purposes, e.g. insulin
production, insect resistance, etc.

 If a protein is not abundant

naturally, its gene can be cloned to
produce the protein in large amounts

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References
:
 Gene biotechnology by S. N. Jogdand.
 Biotechnology by U. Satyanarayana.
 Molecular Biology of Cell by Bruce
Alberts.
 www.slide world.com.

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