cloning vector
DESCRIPTION
TRANSCRIPT
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A SEMINAR ON A SEMINAR ON GENE CLONING VECTORS
PRESENTED BY PRESENTED BY NASREEN SULTANNASREEN SULTAN
M.Sc BIOTECHNOLOGYM.Sc BIOTECHNOLOGY 33RDRD SEMESTER SEMESTER
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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
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Characteristics
It should be able to replicate autonomously.Origin of replication. Selectable markers. Restriction sites.
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TypesTypes
Plasmid as vector .
Bacteriophage as vector.
Cosmid as vector
Phagemid as vector.
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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
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pUC Plasmids
Contains
Ampicillin resistance gene.
Multiple cloning site.
ColEI ( origin ).
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Multiple Cloning Site(MCS or Polylinker)
.
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How insertion takes place
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Screening procedure of cloning vector
Blue/White selection.
replica plating technique.
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Blue/White Selection
Only coloniesfrom bacteria that
have plasmid
IPTG + X-Gal
Overnight growth
Bacteria with plasmid plus insert
Colonies with insert - whiteColonies w/o insert - blue
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Based on the enzymatic reaction of β-galactosidase
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Replica Plating Technique
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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.
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. DNA cloning using phages as vectors
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PHAGE M13 VECTOR
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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.
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Cloning by using Cosmid vectors
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Latest Generation VECTOR: Phagemid- pBluescript
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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.
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THANK YOU
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MODE OF REPLICATION IN PHAGE M13 VECTOR
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