Cloning Vector

40,297 views

Published on

Published in: Education, Technology, Spiritual
12 Comments
73 Likes
Statistics
Notes
No Downloads
Views
Total views
40,297
On SlideShare
0
From Embeds
0
Number of Embeds
144
Actions
Shares
0
Downloads
2,293
Comments
12
Likes
73
Embeds 0
No embeds

No notes for slide
  • CTOES
  • 4362 bp
  • How insertion takes place
  • Replica Plating Technique
  • DNA cloning using
  • PHAGE M13 VECTOR
  • THANKYOU
  • MODE OF REPLICATION IN PHAGE M13 VECTOR
  • 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

    ×