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

Vectors may be

• a) Plasmids:- These are autonomously replicating circular extra -chromosomal DNA .
• b) Bacteriophages:- These are viruses infecting bacteria.
• The best known vector is the plasmid vector .
• pBR322 is the first artificial cloning vector developed in 1977 by Bolivar and Rodriguez from E. coli plasmid.
  The following are the features that are required to facilitate cloning into 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

It also controls the copy no. of the linked DNA.

Selectable marker:-

• It helps to select the host cells which contain the vector (transformants) and eliminate the non-transformants.
• Transformation is defined as the procedure by which a piece of DNA is introduced into a bacterial host .
• The genes encoding resistance to antibiotics such ampicillin chloramphenicol tetracycline or kanamycin etc, re useful selectable marker for E.coli normal E coli cells do not carry resistance
• against any of these antibiotics.
• The normal E. coli cells do not carry resistance against any of these antibiotics.

Cloning sites

To link aliene DNA ,the vectors require very few (mostly single) recognition sites for the restriction enzymes.
More than one recognition sites within the vector can complicate the gene cloning as it will generate several fragments.
Ligation of alien DNA can be carried out at a restriction site present in one of the two antibiotic resistance genes.

Vector for cloning genes in plants and animals.

• Vector for cloning genes in plants is Agrobacterium tumefaciens , a pathogen of server dicot plants which delivers a piece of DNA known as ‘T-DNA ‘to transform normal plant cells into a tumor and direct these tumour cells to produce the chemicals required by the pathogen .
• The tumor inducing (Ti) plasmid of agrobacterium tumefaciens has now been modified into a cloning vector.
  Tumor formation by agrobacterium tumefaciens
• Vector for cloning genes in animals is retrovirus which transforms normal cells into cancerous cells.
• Retroviruses have been disarmed and used to deliver desirable gene.