Cloning Vector
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The document discusses different types of gene cloning vectors including plasmids, bacteriophages, cosmids, and phagemids. Plasmid vectors like pBR322 contain selectable markers and a multiple cloning site. Bacteriophage vectors like lambda phage infect bacteria and can carry larger DNA inserts. Cosmid vectors combine properties of plasmids and phages to clone fragments up to 50kb. All of these vector types allow cloning and replication of foreign DNA fragments in host cells.
Cloning Vector
- 1. A SEMINAR ON GENE CLONING VECTORS PRESENTED BY NASREEN SULTAN M.Sc BIOTECHNOLOGY 3 RD SEMESTER
- 2. INTRODUCTION 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. Characteristics It should be able to replicate autonomously. Origin of replication. Selectable markers. Restriction sites.
- 4. Types Plasmid as vector . Bacteriophage as vector. Cosmid as vector Phagemid as vector.
- 5. Plasmid Vector: pBR322 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. pUC Plasmids Contains Ampicillin resistance gene. Multiple cloning site. ColEI ( origin ).
- 7. Multiple Cloning Site (MCS or Polylinker) .
- 8. How insertion takes place
- 9. Screening procedure of cloning vector Blue/White selection. replica plating technique.
- 10. 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
- 11. Based on the enzymatic reaction of β -galactosidase
- 13. BACTERIOPHAGE VECTORS It infects bacteria. Follow either lytic or lysogenic cycle. Lambda phage vector 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. Size is 48,502 bp.
- 14. . DNA cloning using phages as vectors
- 15. PHAGE M13 VECTOR
- 16. Cosmid vector Combine parts of the lambda chromosome with parts of plasmids. an origin of replication (ori). a cos site(a sequence yield cohesive end) . an ampicillin resistance gene ( amp ), restriction sites for cloning Cosmids can carry up to 50 kb of inserted DNA.
- 17. Cloning by using Cosmid vectors
- 18. Latest Generation VECTOR: Phagemid- p Bluescript
- 19. APPLICATION 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.
- 20. THANK YOU
- 21. MODE OF REPLICATION IN PHAGE M13 VECTOR