Insertional Inactivation

What Is Meant By Insertional Inactivation?

Insertional inactivation is a technique used in recombinant DNA technology. In this procedure, a bacteria carrying recombinant plasmids or a fragment of foreign DNA is made to insert into a restriction site inside a gene to resist antibiotics, hence causing the gene to turn non-functional or in an inactivated state.

An example of this is a pUC19 plasmid vector in which the lacZ gene that encodes for beta-galactosidase can no longer be produced upon the insertion of a foreign gene. Selection or screening is a fundamental process in which once the recombinant DNA is inserted into a particular host cell, it becomes necessary to detect the cells that have received the recombinant or foreign DNA molecules.

This process is referred to as screening or selection. It is based on non-expression or expression of certain traits or characteristics. Insertional inactivation is an effective method of screening. In this procedure, one of the genetic characteristics is disturbed by the introduction of foreign DNA. One of the most influential selection methods of recombinant plasmid for the insertional inactivation procedure is a method known as ‘Blue-white’ selection method.

In this procedure, the lacZ gene which is a reporter gene is inserted in the vector. The enzyme β-galactosidase encoded by the lacZ gene comprises a few recognition sites for restriction enzymes. The β-galactosidase enzyme splits a synthetic substrate X-gal, which is an organic compound abbreviated as BCIG (5-bromo-4-chloro-indolyl-β-D-galactopyranoside) into an insoluble product that is blue in colour.

If a foreign gene is introduced into lacZ, the gene will be deactivated. Hence no blue colour will develop as β-galactosidase is not produced due to deactivation of the lacZ gene. Consequently, the host cell comprising the rDNA will create white coloured colonies on the medium containing X-gal, whereas other cells bearing non-recombinant DNA will tend to develop blue coloured colonies. The recombinants are thus selected on the basis of the colour of the colony. For more information on recombinant DNA technology and related topics, please register at BYJU’S.

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