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Gene Cloning
Dr. Sandeep Agrawal MD
Senior Resident & PhD Scholar
Department of Biochemistry
AIIMS, New Delhi
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Introduced the term “plasmid”

Joshua Lederberg

Invented the technique of DNA cloning in 1973

Stanley Cohen Herbert Boyer Paul Berg

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What is gene cloning? 4

After a large number of cell divisions,

a colony, or clone, of identical host
cells is produced. Each cell in the
clone contains one or more copies of
the recombinant DNA molecule; the
gene carried by the recombinant
molecule is now said to be cloned.
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Restriction endonucleases
Restriction endonucleases 6
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Restriction endonucleases
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Restriction endonucleases

Isoschizomers: Restriction enzymes with same sequence specificity and cut site

Neoschizomers: Enzymes that recognise the same sequence but cleave at different points

Star activity: under certain conditions like elevated pH or low ionic strength, RE are capable
of cleaving sequences which are similar but not identical to their defined recognition sequence
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Restriction endonucleases
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Restriction endonucleases
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Restriction endonucleases
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Restriction digestion
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Restriction digestion
 Temperature: 37oC
 pH: 7.4
 Ionic strength: NaCl & Mg+2 concentration
 Dithiothreitol (DTT): stabilizes the enzyme and prevents its inactivation

 One unit of enzyme activity: The amount of restriction enzyme in

microliters (µL) needed to completely digest (cleave) one microgram
(µg) of substrate DNA in one hour at the optimal temperature of the
enzyme in a 50-µL reaction volume.

 Stopping RE activity:
 Short incubation at 70oC
 Phenol
 EDTA
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Quality control

Ligation can occur only if the 3′

and 5′ termini are left intact, and
only those molecules with a
perfectly restored recognition site
can be recleaved.

A normal banding pattern after

cleavage indicates that both the 3′
and 5′ termini are intact and the
enzyme preparation is free of
detectable exonucleases and
phosphatases
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DNA modifying enzymes
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DNA ligation
 DNA ligases: usually purified from E.coli bacteria that have been
infected with T4 phage. Discontinuity repair.

Sticky ends increase the efficiency of

ligation
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Linkers and Adaptors
 Linkers: short
pieces of double
stranded DNA,
of known
nucleotide
sequence, that
are synthesised
in the test tube. It
is blunt ended
but contains a
restriction site.
 Problem with
linkers:
restriction sites
within DNA of
interest
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Linkers and Adaptors
 Adaptors: Short
synthetic
oligonucleotides
containing one
sticky end

 Problem with
adaptors: two
adaptors could
ligate to one
another.
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Linkers and Adaptors
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Vectors: vehicles for DNA
Desirable properties of a DNA vector:

1. Can independently replicate themselves and the foreign DNA

segments they carry;

2. contain a number of unique restriction endonuclease cleavage

sites that are present only once in the vector;

3. carry a selectable marker (usually in the form of antibiotic

resistance genes or genes for enzymes missing in the host cell) to
distinguish host cells that carry vectors from host cells that do not
contain a vector;

4. are relatively easy to recover from the host cell.

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Vectors: vehicles for DNA
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Plasmid
 Plasmids are naturally occurring replicons in prokaryotic cells and, are stably
inherited in an extrachromosomal state.

 Most plasmids exist as double stranded circular molecules.

 Multiple copies per cell (relaxed plasmids) or limited number of copies per
cell (stringent plasmids).

 Plasmid vectors are ≈1 to 1000 kilo base pairs in size.

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Plasmid as a cloning vector
 Contain replication origin (ori) sequence.

 The ability to survive in normally toxic concentrations of antibiotics such as

chloramphenicol or ampicillin is often due to the presence in the bacterium of
a plasmid carrying antibiotic resistance genes. In the laboratory, antibiotic
resistance is often used as a selectable marker to ensure that bacteria in a
culture contain a particular plasmid.

 Plasmid vectors are modified to contain a multiple cloning site (also called
the polylinker region) which has a number of unique target sites for
restriction endonucleases.
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Plasmid as a cloning vector
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Plasmid as a cloning vector
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Plasmid as a cloning vector
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pBR322

R1 R6-5
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pUC8/pUC18
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pGEM3Z
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Transformation
 Uptake of DNA by bacterial cells is called as transformation.
 Most species of bacteria, including E.coli, take up only limited amounts of DNA
under normal circumstances.
 In order to transform these species efficiently, the bacteria have to undergo some
form of physical and/or chemical treatment that enhances their ability to take up
DNA from the medium in which they grow. Cells that have undergone this
treatment are said to be competent.
 The ice cold calcium chloride affects the cell wall and may also be responsible for
binding DNA to the cell surface. The actual uptake of DNA is stimulated by the
brief heat shock.
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1. Since bacterial species use a restriction-

modification system to degrade foreign DNA
lacking the appropriate methylation pattern,
including plasmids, then why don’t the
transformed bacteria degrade the foreign DNA?

2. Other methods of transformation?

3. Lipofection?

4. Transfection?
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Transformation
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Insertional inactivation
 Insertion of a DNA fragment into the plasmid destroys the integrity of one of
the genes present on the molecule. Recombinants can therefore be identified
because the characteristic coded by the inactivated gene is no longer displayed
by the host cells
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Blue white screening
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Common cloning strains of E.coli

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How to read E.coli genotype
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Growth media
2XYT (1Litre)
 Bacto Tryptone: 10g (supplies amino acids and small peptides)
 Yeast extract: 16g (supplies nitrogen, sugars, inorganic and organic
nutrients)
 NaCl: 5g (provides suitable osmotic environment for bacterial growth)
 pH: 7.2

 Autoclave same day and store at 2-8oC

 Agar Plates: add 15g/L agar to media

 Ampicillin
 Stock: 100mg/ml
 Working: 0.1mg/ml
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Plasmid purification
 Basic steps in isolation of plasmid DNA

1. A culture of bacteria is grown and then harvested

2. The cells are then broken open to release their contents

3. This cell extract is treated to remove all components except the DNA

4. Purification of plasmid DNA from total cell DNA on the basis of:
 Size
 Conformation
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Cell disruption: chemical lysis
 Lysozyme: digests polymeric compounds that give the cell wall its rigidity.
 EDTA: removes magnesium ions that are essential for preserving the overall
structure of the cell envelope, and also inhibits cellular enzymes that could
degrade DNA.
 Detergent (SDS/Triton X-100): aid the process of lysis by removing lipid
molecules and thereby cause disruption of the cell membrane.
 Components such as partially digested cell wall fractions can be pelleted by
centrifugation, leaving the cell extract as a reasonably clear supernatant.
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Plasmid purification on the basis of size

 If the cells are lysed under very carefully controlled conditions, only a minimal
amount of chromosomal DNA breakage occurs. The resulting DNA fragments can
be removed with the cell debris by centrifugation.

 Treatment with EDTA and lysozyme is carried out in the presence of sucrose,
which prevents the cells from bursting immediately. Instead, sphaeroplasts are
formed.

 Cell lysis is now induced by adding a non-ionic detergent such as Triton X-100
(ionic detergents, such as SDS, cause chromosomal breakage).

 This method causes very little breakage of the bacterial DNA, so centrifugation
leaves a cleared lysate, consisting almost entirely of plasmid DNA.
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 Plasmid purification on the basis of
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conformation

1. Alkaline denaturation
method
2. EtBr-CsCl density
gradient centrifugation
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Alkaline denaturation method
 This method makes use of the observation that there is a narrow range of pH
(12-12.5) within which denaturation of linear DNA, but not covalently closed
circular DNA, occurs.

 Chromosomal DNA remains in a high molecular weight form but is denatured.

 Upon neutralization with acidic sodium acetate, the chromosomal DNA

renatures and aggregates to form an insoluble network.

 The high concentration of sodium acetate causes precipitation of protein-SDS

complexes and high molecular weight RNA.
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Alkaline denaturation method

 Provided the pH of the alkaline denaturation step has been carefully controlled,
the CCC plasmid DNA molecules will remain in a native state and in solution,
while the contaminating macromolecules co-precipitate.

 The precipitate can be removed by centrifugation and the plasmid concentrated

by ethanol precipitation.

 If necessary, the plasmid DNA can be purified further by gel filtration.

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Alkaline denaturation method

 Commercially available kits take advantage of the benefits of alkaline lysis and
have as their starting-point the cleared lysate.
 The plasmid DNA is selectively bound to an ion-exchange material, prepacked in
columns or tubes, in the presence of a chaotropic agent (e.g. guanidinium
hydrochloride).
 After washing away the contaminants, the purified plasmid is eluted in a small
volume.
 These kits improve the yield and purity of plasmid DNA.
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EtBr-CsCl density gradient centrifugation

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Plasmid preparation kits by size
 Minipreparation: rapid, small-scale isolation of plasmid DNA from bacteria. A
typical plasmid DNA yield of a miniprep is 50 to 100 µg.

 Midipreparation:The starting E. coli culture volume is 15-25 mL of lysogeny

broth (LB) and the expected DNA yield is 100-350 µg.

 Maxipreparation: The starting E. coli culture volume is 100-200 mL of LB and

the expected DNA yield is 500-850 µg.

 Megapreparation: The starting E. coli culture volume is 500 mL – 2.5 L of LB

and the expected DNA yield is 1.5-2.5 mg.

 Gigapreparation: The starting E. coli culture volume is 2.5-5 L of LB and the

expected DNA yield is 7.5–10 mg.
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If PCR is so much better, then why at all we need to perform gene cloning experiments ?
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References & further reading
 Gene Cloning and DNA Analysis: An Introduction. 6th edition. By T.A. Brown.

 Principles Of Gene Manipulation. 6th edition. By Sandy B. Primrose, Richard M.

Twyman and Robert W. Old.

 Molecular Cloning: A Laboratory manual. 3rd edition. By J. Sambrook, and

David W. Russell.
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