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Cloning Vector

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Cloning Vector

  1. 1. A SEMINAR ON GENE CLONING VECTORS PRESENTED BY NASREEN SULTAN M.Sc BIOTECHNOLOGY 3 RD SEMESTER
  2. 2. INTRODUCTION <ul><li>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. </li></ul><ul><li>Examples: Plasmids,phage or virus </li></ul>
  3. 3. Characteristics <ul><li>It should be able to replicate autonomously. </li></ul><ul><li>Origin of replication. </li></ul><ul><li>Selectable markers. </li></ul><ul><li>Restriction sites. </li></ul>
  4. 4. Types <ul><li>Plasmid as vector . </li></ul><ul><li>Bacteriophage as vector. </li></ul><ul><li>Cosmid as vector </li></ul><ul><li>Phagemid as vector. </li></ul>
  5. 5. Plasmid Vector: pBR322 <ul><ul><ul><li>Contains: </li></ul></ul></ul><ul><li>Selectable Markers : </li></ul><ul><li>Ampicillin resistance gene. </li></ul><ul><li>Tetracycline resistance gene. </li></ul><ul><li>Col E I replication origin. </li></ul><ul><li>Eco RI site. </li></ul>Structure of E.Coli plasmid cloning vector pBR322 4362 bp
  6. 6. pUC Plasmids <ul><ul><li>Contains </li></ul></ul><ul><ul><li>Ampicillin resistance gene. </li></ul></ul><ul><ul><li>Multiple cloning site. </li></ul></ul><ul><ul><li>ColEI ( origin ). </li></ul></ul>
  7. 7. Multiple Cloning Site (MCS or Polylinker) .
  8. 8. How insertion takes place
  9. 9. Screening procedure of cloning vector <ul><li>Blue/White selection. </li></ul><ul><li>replica plating technique. </li></ul>
  10. 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. 11. Based on the enzymatic reaction of β -galactosidase
  12. 12. Replica Plating Technique
  13. 13. BACTERIOPHAGE VECTORS <ul><li>It infects bacteria. </li></ul><ul><li>Follow either lytic or lysogenic cycle. </li></ul><ul><li>Lambda phage vector </li></ul><ul><li>high transformation efficiency, about 1000 times more efficient than the plasmid vector. </li></ul><ul><li>Origin of replication. </li></ul><ul><li>genes for head and tail protein. </li></ul><ul><li>single- stranded protruding cohesive ends. </li></ul><ul><li>Size is 48,502 bp. </li></ul>
  14. 14. . DNA cloning using phages as vectors
  15. 15. PHAGE M13 VECTOR
  16. 16. Cosmid vector <ul><li>Combine parts of the lambda chromosome with parts of plasmids. </li></ul><ul><li>an origin of replication (ori). </li></ul><ul><li>a cos site(a sequence yield cohesive end) . </li></ul><ul><li>an ampicillin resistance gene ( amp ), </li></ul><ul><li>restriction sites for cloning </li></ul><ul><li>Cosmids can carry up to 50 kb of inserted DNA. </li></ul>
  17. 17. Cloning by using Cosmid vectors
  18. 18. Latest Generation VECTOR: Phagemid- p Bluescript
  19. 19. APPLICATION <ul><li>A particular gene can be isolated and its nucleotide sequence determined </li></ul><ul><li>Control sequences of DNA can be identified & analyzed </li></ul><ul><li>Protein/enzyme/RNA function can be investigated </li></ul><ul><li>Mutations can be identified, e.g. gene defects related to specific diseases </li></ul><ul><li>Organisms can be ‘engineered’ for specific purposes, e.g. insulin production. </li></ul>
  20. 20. THANK YOU
  21. 21. MODE OF REPLICATION IN PHAGE M13 VECTOR

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