Cloning involves making genetically identical copies of organisms or genes. Cloning organisms produces clones through asexual reproduction or laboratory techniques. Dolly the sheep was the first mammal cloned in 1997 using nuclei from udder cells implanted into egg cells. Gene cloning involves using restriction enzymes to cut out a target gene, insert it into a bacterial plasmid, and replicate it in bacteria. Common cloning vectors like plasmids and bacteriophages are used to transport cloned sequences between hosts due to characteristics like self-replication and selectable markers. DNA ligase joins DNA strands during cloning, and gel electrophoresis separates cloned DNA fragments by size.

1. Cloning
Direct by

2. Cloning
• Organisms that are genetically identical are
clones
• Asexual Reproduction always produces
clones
• Laboratory Techniques have been
developed that have allowed this to happen
in Animals

3. Cloning in Plants
Plant Cloning

4. Cloning in Animals
• 1950’s first experiments done with
Frogs.
• Early 1990’s some success found with
Mice, Mice were cloned by using
nuclei of cells taken from Mice
Embryos.
• Wilmut et al. Produced “Dolly” in
1997.

6. Cloning – Steps of Dolly
• Used Genetic Information Taken from Udder of Adult
Sheep (donor).
• Unfertilized Eggs Collected from sheep and had
nuclei removed.
• Nuclei from Udder Cells Transplanted into the egg
cells.
• Resulting Cells were cultured and a few began to
divide.
• These early embryos implanted into a surrogate
mother. One of these developed into a Lamb – Dolly.
• DNA Tests confirmed that the Lamb was identical
Genetically to the Sheep that had provided the Udder
Cells.

8. Cloning Genes
• Gene cloning: amplifying a
specific piece of DNA via a
bacteria.

9. Gene Cloning Steps
1. The target gene is isolated and cut out
using a Restriction Enzyme.
2. The bacterial plasmid is cut open using the
same Restriction Enzyme.
3. DNA ligase (Ligase) the target gene into
the plasmid.
4. Monitoring (gel electrophoresis).
5. Recombinant plasmid is inserted into the
bacterium by transformation.

13. Restriction Enzyme
A restriction enzyme or restriction
endonucleases is an enzyme that cleaves
DNA into fragments at or near specific
recognition sites within the molecule known
as restriction site.

14. Restriction Enzymes
Restriction site

15. Type of Restriction Enzymes
• Type I: Restriction Endonucleases are
single multifunctional enzymes with three
different subunits. They required ATP,
Mg2+ and S-adenosylmethionine for its
activity. Their active cleavage site usually
present at least 1000 bp away from the
host specificity site.
Example: EcoB

16. • Type II: restriction endonucleases are most
common type. Enzymes Recognize a
particular target sequence in a duplex DNA
molecule and break the phosphodiester bond
within, or near. require no cofactor other than
Mg2+ .
Example: EcoRI : GAATTC

17. • Type III : Restriction enzymes are
characterized by their ability to cut DNA in
a specific location known and They
required ATP , Mg2+ and S-
adenosylmethionine for its activity.
Example : EcoP1

18. ‘5GAATTC3’
‘3CTTAAG5’
‘5G AATTC3’
‘3CTTAA G5’
‘5CCCGGG3’
‘3GGGCCC5’
‘5CCC GGG3’
‘3GGG CCC5’

20. CLONING VECTORS
Cloning vectors are DNA molecules that are
used to "transport" cloned sequences between
biological hosts and the test tube.

21. CHARACTERISTICS
1.Self replication, multiple copies.
2.Replication origin site.
3.Selectable marker gene.
4.Small size.
5.Low molecular weight.
6.Easily isolated & purified.
7.Easily isolated into host cell.
8.pass into new cells during cell division.
9.Unique restriction sites to facilitate cloning of
insert DNA.

22. Type of cloning vector
•plasmids
•bacteriophageλ , M13
•Cosmids
•Artificial chromosomes BAC, YAC, MAC
etc.

23. What Determines Choice of Vector
• Insert size
• Vector size
• Restriction size
• Cloning
efficiency
Vector Insert size (kb)
Plasmid < 10 kb
Bacteriophage 3 – 18 kb
Cosmids 23 – 45 kb
BACs ≤ 300 kb
PACs 100 – 300 kb
YACs 100 – 3000kb

24. Plasmid
•Extrachromosomal DNA found in bacteria.
•Close circular DNA molecules, supercoiled.
•Can replicate independent of chromosome.
•Can be transfer to other cells by conjugation.
•Can be integrated into the chromosome.
•plasmids carry genes like Resistance to Antibiotics.
•Number of plasmid per cell - controlled by plasmid itself
High copy number > 100 /cell; low copy number < 20 /cell

25. pBR322
• pBR322 is a plasmid and was one of the
first widely used E.coli cloning vectors.
• The p stands for "plasmid," and BR for
"Bolivar" and "Rodriguez.“
• 15 copies.
• Ampilicin resistance gene
• Tetracycline resistance gene
• Origin of replication (Ori)
• Two selectable markers
(Ampilicin,Tetracycline)

27. DNA ligase is a specific type of enzyme,
a ligase that facilitates the joining
of DNA strands together by catalysing the
formation of a phosphodiester bond . It plays
a role in repairing single-strand breaks in
duplex DNA in living organisms, but some
forms (such as DNA ligase IV) may
specifically repair double-strand breaks. with
DNA ligase creating the final phosphodiester
bond to fully repair the DNA.
DNA ligase

28. DNA Ligase
Chromosome
DNA

31. • Gel electrophoresis is a method for separation and
analysis of macromolecules (DNA, RNA and protein) and
their fragments, based on their size and charge.
• DNA samples are loaded into wells (indentations) at one end
of a gel, and an electric current is applied to pull them
through the gel.
• When a gel is stained with a DNA-binding dye, the DNA
fragments can be seen as bands, each representing a
group of same-sized DNA fragments.
Gel electrophoresis

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