By Sweta Tiwari B.Sc. Final year Semester 5 Sarojini naidu P.G autonomous college , Bhopal

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

It should be able to replicate autonomously.Origin of replication. Selectable markers. Restriction sites.

PlasmidsBacteriophageCosmidsPhagemids

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

How insertion takes place

Blue/White selection.

replica plating technique.

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

Replica Plating Technique

It infects bacteria. Follow either lytic or lysogenic cycle.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.Can take DNA insert upto 15-25 kb.

. DNA cloning using phages as vectors

Combine parts of the lambda chromosome with parts of plasmids.An origin of replication (ori). cos site(a sequence yield cohesive end) ampicillin resistance gene (amp). restriction sites for cloning. Cosmids can carry up to 50 kb of inserted DNA.

Phagemids are shortened linear lambda genomes containing DNA replication and lytic function plus the cohesive ends of the phage.

Their middle non essential segment is replaced by a linearized plasmid with intact replication module .

Can take DNA insert of 50-60 kb. They are packaged into phage particles.

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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