The following points highlight the four main features that are required to facilitate cloning into a vector. The features are: 1. Origin of Replication 2. Selectable Marker 3. Cloning (Recognition) Sites 4. Vectors for Cloning Genes in Plants and Animals.
Feature # 1. Origin of Replication (Ori):
This is a sequence from where replication starts and any piece of DNA, when linked to this sequence can be made to replicate within the host cells.
This sequence is also responsible for controlling the copy number of the linked DNA.
Feature # 2. Selectable Marker:
It helps in identifying or selecting transformants and eliminating non-transformants; and selectively permits the growth of the transformants. Transformation is a procedure, through which a piece of DNA is introduced in a host bacterium. Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline, kanamycin, etc., are considered as useful selectable markers for E. coli.
The normal E. coli do not carry resistance against any of these antibiotics.
Feature # 3. Cloning (Recognition) Sites:
These are generally required to link the foreign or alien DNA with the vector. For this, the vector requires very few or single recognition sites for commonly used restriction enzymes. If more than one recognition sites is present within the vector, it will generate several fragments that will lead to more complication in gene cloning. The ligation of the foreign DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.
Selection of recombinants formed can be done by one of the following methods, given below:
(a) Inactivation of Antibiotics:
If a foreign DNA is ligated at the Bam HI site of tetracycline-resistance gene in the vector pBR322, the recombinant plasmid will lose tetracycline resistance. But, it can still be selected out from non-recombinant ones by plating transformants on ampicillin containing medium. The transformants growing on medium containing ampicillin are then transferred on a medium containing tetracycline.
The recombinants will grow on ampicillin containing medium but not on that containing tetracycline. On the other side, non-recombinants will grow on both media and thus, recombinants are selected. In this example, one antibiotic resistance gene helps in selecting the transformants whereas, the other antibiotic resistance gene gets inactivated due to insertion of alien DNA and helps in selection of recombinants.
It is a cumbersome procedure. Therefore, the alternative selectable markers have been developed, which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of chromogenic substrate.
(b) Insertional Inactivation:
The recombinants and non-recombinants can also be differentiated on the basis of colour production in presence of chromogenic substrate. Here, a recombinant DNA is inserted within the coding sequence of an enzyme β -galactosidase, which results into inactivation of the enzyme. Therefore, the bacterial colonies having inserted plasmid, shows no colouration, while those without plasmid will show blue colour.
Feature # 4. Vectors for Cloning Genes in Plants and Animals:
In plants, the tumour inducing (Ti) plasmid of Agrobacterium tumefaciens (a pathogens of several dicot plants) is used as a cloning vector.
(a) It delivers a piece of DNA known as tDNA in the Ti plasmid, which transforms a normal plant cell into tumour cells to produce chemicals against pathogens.
(b) Similarly, retro virus, adeno virus, papilloma virus are also now used as cloning vectors in animals because of their ability to transform normal cells into cancerous cells.