pharmacology

1. CLONING OF DISEASE
GENE
NAME – BHARAT KUMAR
ROLL NO. - 17MPH202

2. WHAT IS CLONING
From the Greek - klon, a twig
A clone is a genetically identical copy of an organism, and it may be naturally
occurring or created in the lab. Through the process of asexual reproduction,
organisms such as bacteria (and some plants) create offspring that are
genetically identical to the parent. Modern genetic technology can also be used
to create clones.

3. Types Of Cloning
• GENE CLONING
• REPRODUCTIVE CLONING
• THERAPEUTIC CLONING

4. GENE CLONING
• Making multiple copies of a single gene
• 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.

5. GENE CLONING

6. BASIC STEPS OF GENE CLONING
Construction of recombinant DNA molecule
Transport of the recombinant DNA to the host cell
Multiplication of recombinant DNA molecule
Division of the host cell
Numerous cell division resulting in a clone

7. Gene cloning requires specialized tools and
techniques
Vehicles: The central component of a gene cloning experiment is the vehicle,
which transport the gene into the host cell and is responsible for its replication.
To act as a cloning vehicle a DNA molecule must be capable of entering a host
cell and, once inside, replicating to produce multiple copies of itself.
Vector: A DNA molecule, capable of replication in a host organism, into
which a gene is inserted to construct a recombinant DNA molecule.

8. Characteristic of a Vector
• It must be able to replicate
• There must be some way to introduce vector DNA into a cell
• There must be some means of detecting its presence, preferably by plating
test in petri dishes

9. PLASMID
Characteristics:
• They are small and contain 3-5 kb of DNA
• They are contain a suitable markers (antibiotic resistant)
• They contain suitable restriction sites that can be used for inserting DNA
fragments for cloning
Bacterium
Chromosomal DNA
Plasmid DNA

10. CLONING VECTOR
• A plasmid into which the gene of interest
is introduced
• Contains a number of specific genes useful
in selection

11. CLONING VECTOR COMPONENTS
• Ori
• ampR gene
• lacZ gene
• Restriction gene

12. Activity : Gene Cloning
• In this activity you will determine the function of the ampR and lacZ gene
and explain its use in gene cloning

13. Cloning vector components
• Replication origin (ori) :
Allows plasmid to replicate in the
host cell

14. Cloning vector components
• Antibiotic resistance (ampR) gene :
allows cells to be resistance to
ampicillin (an antibiotic)
• Selection for host cells that have
resistance
• Thus, selecting for transformation

15. Cloning vector components
• β-galactosidase (LacZ) gene :
enzyme produced will change a
clear substrate called X-gal into a
blue product

16. β-galactosidase Reaction
• β-gal acts on X-Gal (a clear soluble substrate) to produce a blue precipitate

17. Step 1: Forming Recombinant DNA
• Where would you insert the DNA of
interest so that you can “see” it in the
bacterial cell (assume cells are grown in
X-gal)?

18. Step 1: Forming Recombinant DNA
• Ligate the gene of interest
into the vector such that it
interrupts the lacZ gene
• Thus β-galactosidase is not
made

19. Step 2: Transformation
• Transform recombinant DNA into bacterial
cell
• As bacterial cells multiply, the gene of
interest will be replicated with each cell

20. Step 2: Transformation
• Bacteria grown in flasks of liquid
medium
• Incubate at optimal growing
temperature

21. Step 3: Selection
• Selection: identify colonies of bacteria containing the recombinant DNA
with gene of interest
• Possible bacterial clone products:
A. bacteria without vector
B. bacteria with vector without gene
C. bacteria with vector with the gene of interest

22. Step 3: Selection
• Plating: taking a sample of the
bacteria and growing them on plates
• Plates have a medium containing:
antibiotics
X-gal

23. Selection mechanism: Antibiotic resistance
• Select for bacterial clones that
contain a vector (select for proper
transformation)
• Bacteria are grown on Petri plate
containing a specific antibiotic (e.g.
ampicillin)

24. Antibiotic Resistance
• Vector confers antibiotic resistant to
bacteria because the vector contains an
ampR gene
• Only bacterial cells that properly
transformed the vector will live and grow
on the plate

25. Selection for successful transformation

26. Selection Mechanisms: β-galactosidase
Screening
• Select for bacterial clones that contain a
vector with gene of interest (select for
proper ligation)
• Bacteria are grown on Petri plates
containing X-Gal

27. Possible Transformation Results
LB Medium additions No vector Cloning vector Recombinant DNA
Amp No growth White White
X-gal White Blue White
Amp + X-gal No growth Blue White

29. Cloning Application: Flavr savr Tomatoes
• First genetically modified produce
• Genetically modified tomatoes that suppressed a gene responsible for fruit
ripening
• Process required cloning the gene and transforming a reverse orientation
copy which would have inhibitory effects.

30. Cloning Application: Bt Plants
• Bacillus Thuringiensis
a bacterium used a
biological pesticide
• Bt gene is cloned into
plants so that they will
be resistant to pests.

31. THANK YOU
.