Cloning vectors

1. By
Sweta Tiwari
B.Sc. Final year Semester 5
Sarojini naidu P.G autonomous college ,
Bhopal

2.  A cloning vector is a DNA molecule in which foreign
DNA can be inserted or integrated and which is
further capable of replicating within host cell to
produce multiple clones of recombinant DNA.
 Examples: Plasmids,phage or virus

3. It should be able to
replicate
autonomously.
Origin of replication.
Selectable markers.
Restriction sites.

4. Plasmids
Bacteriophage
Cosmids
Phagemids

5. Contains:
• Selectable Markers:
Ampicillin resistance gene.
Tetracycline resistance gene.
Col E I replication origin.
Eco RI site.
Structure of E.Coli plasmid cloning vector pBR322
4362 bp

6. How insertion takes place

7. Blue/White selection.
replica plating technique.

8. Blue/White Selection
Only colonies
from bacteria that
have plasmid
IPTG + X-Gal
Overnight growth
Bacteria with
plasmid plus insert
Colonies with insert - white
Colonies w/o insert - blue

9. Replica Plating Technique

10. It infects bacteria.
Follow either lytic or lysogenic cycle.
high transformation efficiency, about 1000
times more efficient than the plasmid vector.
Origin of replication.
genes for head and tail protein.
single- stranded protruding cohesive ends.
Can take DNA insert upto 15-25 kb.

11. .
DNA cloning using phages as vectors

12. Combine parts of the lambda chromosome
with parts of plasmids.
An origin of replication (ori).
cos site(a sequence yield cohesive end)
ampicillin resistance gene (amp).
restriction sites for cloning.
Cosmids can carry up to 50 kb of inserted
DNA.

13.  Phagemids are shortened linear lambda
genomes containing DNA replication and lytic
function plus the cohesive ends of the phage.
 Their middle non essential segment is replaced
by a linearized plasmid with intact replication
module .
 Can take DNA insert of 50-60 kb.
 They are packaged into phage particles.

14.  A particular gene can be isolated and its nucleotide
sequence determined
 Control sequences of DNA can be identified &
analyzed
 Protein/enzyme/RNA function can be investigated
 Mutations can be identified, e.g. gene defects
related to specific diseases
 Organisms can be ‘engineered’ for specific
purposes, e.g. insulin production.

15. THANK YOU