Which method uses a chromogenic substrate to distinguish recombinant…

The correct answer is Option (2) → Insertional inactivation of the β-galactosidase gene

After a foreign gene is put into a plasmid and the plasmid into bacteria, the recombinant colonies (plasmid with the insert) must be told apart from the non-recombinant ones. One method uses a chromogenic substrate, a substance that gives a colour when an enzyme acts on it.

Option (1) Selection on plates containing two different antibiotics — Incorrect. Plating on two antibiotics also picks out recombinants, through inactivation of an antibiotic resistance gene. But it uses antibiotics, not a chromogenic substrate, and the book calls it cumbersome.

Option (2) Insertional inactivation of the β-galactosidase gene — Correct. Insertional inactivation of the β-galactosidase gene is the method that uses a chromogenic substrate: recombinant colonies stay colourless, non-recombinant colonies turn blue.

Option (3) Treatment of host cells with a divalent cation such as calcium — Incorrect. Treatment with a divalent cation such as calcium makes bacterial cells competent to take up DNA. It helps DNA enter the cells; it does not tell recombinants apart.

Option (4) Heat shock at 42°C — Incorrect. Heat shock at 42°C is the step that forces recombinant DNA into competent bacterial cells. It is part of transformation, not selection.

NCERT Extract:
Cloning Vectors – “… Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure because it requires simultaneous plating on two plates having different antibiotics. Therefore, 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 a chromogenic substrate. In this, a recombinant DNA is inserted within the coding sequence of an enzyme, β-galactosidase. This results into inactivation of the gene for synthesis of this enzyme, which is referred to as insertional inactivation. The presence of a chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the β-galactosidase gene and the colonies do not produce any colour, these are identified as recombinant colonies.”