A SEMINAR ON A SEMINAR ON GENE CLONING VECTORS

PRESENTED BY PRESENTED BY NASREEN SULTANNASREEN SULTAN

M.Sc BIOTECHNOLOGYM.Sc BIOTECHNOLOGY 33RDRD SEMESTER 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.

TypesTypes

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 coloniesfrom bacteria that

have plasmid

IPTG + X-Gal

Overnight growth

Bacteria with plasmid plus insert

Colonies with insert - whiteColonies 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- pBluescript

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