BIOTECH 3

 

CLONING VECTORS

• A cloning vector is a small piece of DNA, taken from any organism into which a foreign DNA fragment can be inserted for cloning purposes.
• Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.
• If an alien piece of DNA with bacteriophage or plasmid DNA, we can multiply its numbers equal to the copy number of the plasmid or bacteriophage.

The following are the features that are required to facilitate cloning into a vector are-

• Origin of replication (ori)
• Selectable marker
• Cloning sites

Origin of replication (ori)- This is the sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells

Selectable marker-

• It helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants.
• Transformation is a procedure through which a piece of DNA is introduced in a host bacterium.
• The genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc., are useful selectable markers for E. coli as the normal E. coli cells do not carry resistance against any of these antibiotics.
• Antibiotic resistance genes help in selecting recombinants from non-recombinants by a method called insertional inactivation where a recombinant DNA is inserted within the coding sequence of an enzyme β-galactosidase in the presence of a chromogenic substrate which results into inactivation of the enzyme.
• The presence of a chromogenic substrate gives blue colored colonies if the plasmid in the bacteria does not have an insert.
• Presence of insert results into insertional inactivation of the β-galactosidase and the colonies do not produce any colour, these are identified as recombinant colonies.

Cloning sites-

• Cloning sites are the recognition sites of the restriction enzymes.
• The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.

For example- ligation of a foreign DNA at the Bam H I site of tetracycline resistance gene in the vector pBR322

• Vector for cloning genes in plants is Agrobacterioum tumifaciens, a pathogen of several dicot plants which delivers a piece of DNA known as ‘T-DNA’ to transform normal plant cells into a tumor and direct these tumor cells to produce the chemicals required by the pathogen.
• The tumor inducing (Ti) plasmid of Agrobacterium tumifaciens has now been modified into a cloning vector.

• Vector for cloning genes in animals is retrovirus which transforms normal cells into cancerous cells.
• Retroviruses have been disarmed and used to deliver desirable genes into animal cells