Gene cloning presentation Gene Cloning: The insertion of a fragment of DNA carrying a gene into a cloning vector and subsequent propagation of recombinant DNA molecules into many copies is known as gene cloning.

2. Gene Cloning: The insertion of a fragment of DNA
carrying a gene into a cloning vector and subsequent
propagation of recombinant DNA molecules into
many copies is known as gene cloning.
Gene Cloning:

3. •Gene cloning involves using bacteria to make multiple
copies of a gene
•Foreign DNA is inserted into a plasmid, and the
recombinant plasmid is inserted into a bacterial cell
•Reproduction in the bacterial cell results in cloning of
the plasmid including the foreign DNA
•This results in the production of multiple copies of a
single gene

4. Gene Manipulation:
Gene manipulation is defined as the formation
of a new combination of a heritable material by
the insertion of nucleic acid molecules (DNA
molecules) into bacterial plasmid or any other
vector so as to allow their incorporation in to
the host organism, in which they are capable of
containing propagation.

5. Figure 20.2
Bacterium
Bacterial
chromosome
Plasmid
2
1
3
4
Gene inserted into
plasmid
Cell containing gene
of interest
Recombinant
DNA (plasmid)
Gene of
interest
Plasmid put into
bacterial cell
DNA of
chromosome
(“foreign” DNA)
Recombinant
bacterium
Host cell grown in culture to
form a clone of cells containing
the “cloned” gene of interest
Gene of
interest
Protein expressed from
gene of interest
Protein harvestedCopies of gene
Basic research
and various
applications
Basic
research
on protein
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Human growth
hormone treats
stunted growth

6. Gene Cloning Technique

8. •Transformation:
• A bacterium takes up DNA from the medium.
• Recombination takes place between introduced genes and the bacterial
chromosome.
Gene Transfer in Bacteria

9. Transformation
•Methods:
•Electroporation
•Freeze Thaw Method
•Calcium Chloride Mediated
Transformation
•Calcium Phosphate Mediated
Transformation

10. Calcium Chloride Method
•Treatment with calcium
chloride in the early log phase
of growth for Competence
•Bacterial cell membrane is
permeable to chloride ions, but
is non-permeable to calcium
ions
Chloride
Ions
E.Coli

11. Calcium Chloride Method
• As the chloride ions enter the
cell, water molecules
accompany the charged
particle
• Influx of water causes the
cells to swell and is necessary
for the uptake of DNA
• The exact mechanism of this
uptake is unknown.

12. Calcium Chloride Method
• DNA of interest is then added to the cells

13. Calcium Chloride Method
•Calcium chloride treatment
be followed by addition of
DNA of interest then by
heat.
•The heat shock step is
necessary for the uptake of
DNA.

14. Calcium Chloride Method
•Temperatures > 42degC: Bacteria’s ability to uptake
DNA reduces
•Extreme temperatures: Bacteria dies.

15. Calcium Chloride Method
•After the heat shock step intact plasmid DNA
molecules replicate in bacterial host cells
•To help the bacterial cells recover from the
heat shock cells are briefly incubated with non-
selective growth media

16. Calcium Chloride Method
• As the cells recover, plasmid
genes are expressed
• Bacterial colonies selected
using antibiotic selection
techniques

18. screening technique
Lose of antibiotic resistance.
Colony hybridization.
Immuno chemical method.

19. PCR Cloning Method
 PCR cloning differs from traditional cloning in that the DNA fragment of
interest, and even the vector, can be amplified by the Polymerase Chain
Reaction (PCR) and ligated together, without the use of restriction
enzymes.
 PCR cloning is a rapid method for cloning genes, and is often used for
projects that require higher throughput than traditional cloning methods
can accommodate.
 It allows for the cloning of DNA fragments that are not available in large
amounts.
 Typically, a PCR reaction is performed to amplify the sequence of
interest, and then it is joined to the vector via a blunt or single-base
overhang ligation prior to transformation.
 Early PCR cloning often used Taq DNA Polymerase to amplify the gene.
This results in a PCR product with a single template-independent base
addition of an adenine (A) residue to the 3' end of the PCR product,
through the normal action of the polymerase.
 These "A-tailed" products are then ligated to a complementary T-tailed
vector using T4 DNA ligase, followed by transformation.

21.  High-fidelity DNA polymerases are also now routinely used to amplify sequences
with the PCR product containing no 3' extensions.
 The blunt-end fragments are joined to a plasmid vector through a typical ligation
reaction or by the action of an "activated" vector that contains a covalently
attached enzyme, typically Topoisomerse I, which facilitates the vector:insert
joining.
 Some PCR cloning systems contain engineered "suicide" vectors that include a
toxic gene into which the PCR product must be successfully ligated to allow
propagation of the strain that takes up the recombinant molecule during
transformation.
 A typical drawback common to many PCR cloning methods is a dedicated vector
that must be used.
 These vectors are typically sold by suppliers, like NEB, in a ready-to-use linearized
format and can add significant expense to the total cost of cloning. Also, the use of
specific vectors restricts the researcher's choice of antibiotic resistance, promoter
identity, fusion partners, and other regulatory elements.

22. Advantages:
High efficiency, with dedicated vectors
Amenable to high throughput
Disadvantages:
Limited vector choices
Higher cost
Lack of sequence control at junction
Multi-fragment cloning is not straight forward
Directional cloning is difficult

23. Thank you