Gene cloning involves making many identical copies of a gene. The basic steps include isolating DNA from an organism, cutting the DNA into fragments using restriction enzymes, inserting the fragments into plasmids, and transforming the plasmids into bacterial cells. Each bacterial cell will contain copies of the gene of interest, allowing scientists to isolate and study that gene. Viruses and bacteria can act as vectors, or carriers, to transfer genes into cells. Plasmids and bacteriophages are commonly used vectors for cloning genes into bacterial cells.

1. UNIT-1
Gene Cloning

2. What is a Gene ?
• Gene is a hereditary unit consisting of a
sequence of DNA that occupies a specific
location on a chromosome and determines a
particular characteristic in an organism.

3. WHAT IS CLONING ?
• Cloning in Biotechnology refers to processes
used to create identical copies of DNA
fragments , cells or organisms .
* Clone is a
group of cells derived from a single
parental cell by asexual reproduction

4. These identical copies are called
clones
Cloning refers to DNA fragments -
molecular or gene cloning
Cloning refers to cells – cell cloning
Cloning refers to organism - organism
cloning

5. What is gene cloning ?
• To "clone a gene" is to
make many copies of it
• Act of making many identical
copies of gene
• Gene can be an exact copy
of a natural gene
• Gene can be an altered
version of a natural gene

6. Whole organisms are cloned too,
but differently

7. BASIC STEPS IN GENE CLONING
• Preparation of Pure
sample of DNA from the
organism of desire
• Cutting of the DNA
molecule using
Restriction enzymes to
form fragments
• Analysis of DNA
fragments using
electrophoresis
The action of a restriction enzyme ,EcoRI

8. Steps in Cloning a Gene
• The First step in cloning a gene is to isolate the
DNA from the organism that contains the
desired gene.

9. Steps in Cloning a Gene
• The isolated DNA is purified and then
fragmented with a restriction enzyme.
• Restriction enzymes used in cloning produce
staggered cuts in specific sequences in the
DNA, generating fragments with the cohesive
ends.

10. Steps in Cloning a Gene
• Each fragment has a single stranded sequence
of nucleotides on its ends that is capable of
hybridizing with DNA that has been
fragmented with the same restriction enzyme.

11. • The DNA fragments are then incorporated into
the plasmids.

12. • The type of plasmid used for cloning
has a single restriction site, and
when cleaved by the restriction
enzyme, generates the same
cohesive ends that are in the
fragments of DNA to be cloned.

13. • The cohesive ends of the plasmid and DNA
fragments now line up and the enzyme, DNA
ligase, is used to form Phosphodiester
bonds.

14. • The next step is to incorporate the
plasmid into bacterial host cells by
transformation.

15. • Each cell contains a different segment of DNA
from the original organism. Taken together
these cells represent a DNA library.

16. • The cells can now be plated out on
agar medium.
• The colony of cells containing the
desired cloned gene can then be
identified and isolated.

18. INFERENCE
• Within the host cell the vector multiplies and
produces numerous identical copies not only of itself
but also of the gene that it carries.
• After that the host cell divides … as the
recombinant vector replicates
• Now 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 is cloned.

19. Application of Bacteria
& Virus used in Genetic
Engineering

20. What is genetic engineering?
• Genetic engineering refers to the direct
manipulation of DNA to alter an organism’s
characteristics in a particular way.
• This may mean changing one base pair (A-T or C-
G), deleting a whole region of DNA, or
introducing an additional copy of a gene.
• It may also mean extracting DNA from another
organism’s genome and combining it with the
DNA of that individual.

22. • A gene is much too small to be inserted with
some kind of microsurgery.
• Therefore some carrier ("vector") is required
which takes the gene into the recipient cell
and somehow gets it inserted into the DNA of
the recipient.

23. Methods for gene insertion
• Different means are used for carrying the desired
gene into the hereditary substance..
• For plants, the most common method is the use
of a bacterium, Agrobacterium tumefaciens.
• For animals, certain viruses are used.

24. 1. Bacteria as gene carriers

25. PLASMIDS
A small circles of DNA found in bacteria and
some other organisms.
• Plasmids can replicate independently of the
host cell .

27. Desirable properties of a Vector
• It should be small.
• Its DNA sequence should be known.
• It should grow to high copy number in the host
cell.
• It should contain a selectable marker that allows
easy selection of transformed host cell.
• It should replicate autonomously
• Should be easy to isolate & purify
• Should contain one unique restriction enzymes.

28. • Also, when expression of DNA is needed then
expression vectors are used.
• Expression vectors should contain control
elements; promoter, operator etc..

29. Types of vectors
• Choice of vector is dependent on insert size
and application.
• For Bacterial Cells:
• Plasmids
• Bacteriophage( Lambda phage & M 13 Phage)
• Cosmids
• Phagemids
• YAC( Yeast Cloning Vectors)

30. For Plant cells:
Binary Vector system
Co -integrative vector system
For Animal/ Mammalian cells:
Retrovirus
SV40 Viral Vector
EBV( Epstein- Barr Virus), BPV( Bovine Papilloma
Virus)

31. Cloning Vector
Contains:
• An origin of replication
• Selectable marker gene ( eg. Ampicillin
resistance gene) and
• Multiple cloning site containing unique
restriction enzymes sites..

32. Plasmid
• A small circular DNA molecule found in
bacteria.
• Used as cloning vectors.
• Example: PUC19

33. Plasmid classification
• Fertility or F – plasmids
• Resistance or R plasmids
• Col plasmids
• Degradative plasmids
• Virulence

34. 2. Bacteriophages
• Phages are very simple in structure,
• Consisting of a DNA molecule carrying a
number of genes, including several for
replication of the phage, surrounded by a
protective coat or capsid made up of protein
molecules

36. • Examples: T2, T4 and T6 Bacteriophages that
infect E.coli.
• Filamentous phages with single stranded DNA
such as M13
• Transfer of genetic elements from one
bacterium to another by a bacteriophage is
termed as transduction.

37. Why is Bacteria used for genetic
engineering?

38. • Generation time is very small that
is 20 minutes.
• Bacteria's DNA is very simple and
can easily be transplanted from
one bacterium to another

39. Cloning Process

40. How are viruses used in
Genetic Engineering
• Viruses are tiny, nonliving particles that must
have a host cell to replicate and flourish.
• Ability to attach to and invade specific cells,
and incorporate the DNA (and/or RNA) they
are carrying into the host cell, where it
combines with the host cell's DNA.