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Feature of Cloning Vectors

Cloning vectors feature an origin of replication to control the copy number of target DNA, selectable markers for identifying transformants, and cloning sites for restriction enzymes. Insertational inactivation allows for the identification of recombinant colonies through gene deactivation or color production. Various transformational techniques, such as heat shock, micro-injection, and gene gun methods, are employed to introduce recombinant DNA into host cells.

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5 views

Feature of Cloning Vectors

Cloning vectors feature an origin of replication to control the copy number of target DNA, selectable markers for identifying transformants, and cloning sites for restriction enzymes. Insertational inactivation allows for the identification of recombinant colonies through gene deactivation or color production. Various transformational techniques, such as heat shock, micro-injection, and gene gun methods, are employed to introduce recombinant DNA into host cells.

Uploaded by

johnkelvinfredrick.pgtiizoo
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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FEATURES OF

CLONING VECTORS
ORIGIN OF REPLICATION (ORI)

• Start site of replication.


• Controls copy number of the linked target dna.
• Higher copy number – more copies of target dna
SELECTABLE MARKER

• Helps in identification and elimination of non transformants.


• Ampicillin, chloramphenicol, tetracyclin resistance.
• Normal E.coli does not posses these genes.
CLONING SITES

• Recognition site for commonly used restriction enzymes.


• Preferably few to a single site: multiple sites produces many fragments
on restriction
• Ligation is carried out at a restriction site of a selectable marker.
INSERTIONAL INACTIVATION

• Correct insertion into resistance gene causes deactivation of the


resistance gene.
• Alternative method – selection on the ability to produce colour in the
presence of a chromogenic substance. Eg; beta galactosidase enzyme
coding gene.
• Helps in identification of recombinant colonies.
VECTORS FOR CLONING GENES IN PLANTS
AND ANIMALS.
• Bacteria and viruses which take over the cellular mechanism are
studied.
• Eg ; Agrobacterium tumifaciens – inserts a piece of dna that transform
normal cells into tumour cells that produce necessary chemicals. (TI
plasmid)
• Eg; retroviruses
• dNA of these viruses are used as carriers for transforming animal cells.
TRANSFORMATIONAL TECHNIQUES TO INPUT
RECOMBINANT DNA INTO THE HOST
• Heat shock method – cell is exposed to bivalent cation like calcium,
cells incubated with rdna in ice followed by heat shock of 42 degrees
and then placed in ice again
• Micro-injection method – micropippetes or micro injection
• Gene gun method or biolistic method – gold or tungsten particles (1-2
nano meter) with rdna.
• Disarmed pathogens method infectious organism neutered and used as
vector.

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