i) The characteristics of a cloning vector are:-
a) Origin of replication (Ori) : It is a sequence which causes the initiation of replication and is also the place to link the foreign piece of DNA.
b) Selectable Marker : It is the site containing antibiotic resistance genes which helps us to identify the transformant and non-transformant vectors after the juncture of new piece of DNA.
c) Recognition Site: It is the cloning site where the piece of foreign DNA is inserted during the formation of Recombinant DNA molecule.
ii) DNA since being a hydrophilic molecule is unable to cross the bacterial cell membrane.
Various techniques used to transfer DNA into the cell are:-
a) By placing the cell in medium with high Ca2+ (divalent) ion concentration, which opens pore in cell membrane and allow entry of DNA into the cell.
b) By giving heat shock treatment i.e. putting the cell on ice and then immediately heated to 42 degree Celcius which opens pores and causes entry of DNA.
c) Micro-injection: Foreign DNA is directly injected into the cell using micro-needles.
d) Electroporation: Electric impulses induce pores on cell membrane through which DNA enters the cell.
e) Direct DNA Injection
f) Gene Gun/Biolistic: Small pellets of gold /tungsten are coated with DNA and hen bombarded on the cell at high velocity.
i) Cutting the desired gene at specific location is done by Restriction enzymes i.e. restriction endonucleases.
They find the specific pallindromic sequence in the gene and cut it at the ends of the pallindromic sequence.
ii) Synthesis of this desired can be done transfering this gene into a bacterial cell for replication by the use of a plasmid.
The desired gene is first selected by electrophoresis and then introduced in the cell for production of multiple copies by the help of a vector i.e. a bacteriophage or a plasmid, etc.
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