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tetracycline or kanamycin, etc, are some useful selectable markers for E. coli.

• Ligation of alien DNA is carried out at a restriction site present in one of the twoantibiotic
resistance genes. Example is ligating a foreign DNA at the Bam HI site of tetracycline
resistance gene in the vector pBR322.
-» The recombinant plasmids will lose tetracycline resistance due to insertion of foreign
DNA. But, it still can be selected out from non-recombinant ones by plating the
transformants on ampicillin containing medium.
-» The transformants growing on ampicillin containing medium are then transferred on a
medium containing tetracycline.
-» The recombinants will grow in ampicillin containing medium but not on that containing
tetracycline.
The non-recombinants will grow on the medium containing both the antibiotics.
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.
* Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure,
so alternative selectable markers are developed which differentiate recombinants from non-
recombinants on the basis of their ability to produce colour in the presence of a
chromogenic substrate.
-» In this method, a recombinant DNA is inserted within the coding sequence of an enzyme
J3-galactosidase.
-» This results into inactivation of the enzyme, P-galactosidase (insertional inactivation).
-> The bacterial colonies whose plasmids do not have an insert, produce blue colour, but
others do not produce any colour, when grown on a chromogenic substrate.
(c) Cloning sites are required to link the alien DNA with the vector.
• The vector requires very few or single recognition sites for the commonly used restriction
enzymes.
• The presence of more than one recognition sites within the vector will generate several
fragments leading to complication in gene cloning.
(d) Vectors for cloning genes in plants and animals are many which are used to clone genes