Botany UG degree

Insertional Inactivation – BlueWhite Colony Selection Method – DNA screening methods

It is a powerful method used for screening of recombinant plasmid. In this method, a reporter gene lacZ is inserted in the vector. The lacZ encodes the enzyme β-galactosidase and contains several recognition sites for restriction enzyme. β-galactosidase breaks a synthetic substrate called X-gal (5-bromo-4-chloro-indolyl-βD-galacto-pyranoside) into an insoluble blue coloured product. If a foreign gene is inserted into lacZ, this gene will be inactivated.

Therefore, no-blue colour will develop (white) because β-galactosidase is not synthesized due to inactivation of lacZ. Therefore, the host cell containing r-DNA form white coloured colonies on the medium contain X-gal, whereas the other cells containing non-recombinant DNA will develop the blue coloured colonies. On the basis of colony colour, the recombinants can be selected.

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