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Cloning Vector
 

Cloning Vector

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  • 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 Cloning Vector Presentation Transcript

  • A SEMINAR ON GENE CLONING VECTORS PRESENTED BY NASREEN SULTAN M.Sc BIOTECHNOLOGY 3 RD SEMESTER
  • 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
  • Characteristics
    • It should be able to replicate autonomously.
    • Origin of replication.
    • Selectable markers.
    • Restriction sites.
  • Types
    • Plasmid as vector .
    • Bacteriophage as vector.
    • Cosmid as vector
    • Phagemid as vector.
  • 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
  • pUC Plasmids
      • Contains
      • Ampicillin resistance gene.
      • Multiple cloning site.
      • ColEI ( origin ).
  • Multiple Cloning Site (MCS or Polylinker) .
  • How insertion takes place
  • Screening procedure of cloning vector
    • Blue/White selection.
    • replica plating technique.
  • 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
  • Based on the enzymatic reaction of β -galactosidase
  • Replica Plating Technique
  • 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.
  • . DNA cloning using phages as vectors
  • PHAGE M13 VECTOR
  • 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.
  • Cloning by using Cosmid vectors
  • Latest Generation VECTOR: Phagemid- p Bluescript
  • 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.
  • THANK YOU
  • MODE OF REPLICATION IN PHAGE M13 VECTOR
  •