. Define Insertional inactivation
- In plasmid for example-pBR322, has a variety of unique cloning sites for restriction endonucleases to digest DNA.
- In ampicillin resistance gene, two cloning sites Pst I and Pvu I are present
- In tetracycline resistance gene, two cloning sites Bam H I and Sal I are present
- In pBR322, some other unique restriction sites are Eco RI, Cla I, HindIII and PvuII, rop codes for the proteins involved in the replication.
- The presence of restriction sites within the markers tetR and ampR permits an easy selection for the cells transformed with the recombinant pBR322.
- DNA fragments are inserted into the plasmid using enzyme PvuI restriction endonuclease, DNA is placed t within the gene ampR, this makes this gene non-functional.
- Bacterial cells containing this plasmid pBR322, will be unable to grow in the presence of ampicillin, but will grow on tetracycline.
- Due to this inactivation of antibiotics, selection of recombinants becomes a very cumbersome process.As, we need to prepare two plates in a replica plating method.
- Another, vector pUC8 is used to differentiate recombinants from non-recombinants on the basis of their ability to produce color in the presence of a chromogenic substance (X-gal).
- In this way recombinant DNA is inserted into the coding sequence of an enzyme β-galactosidase. This causes inactivation of an enzyme, known as insertional inactivation.
In plasmid pBR322 and pUC8, when the gene gets inactivated due to restriction endonuclease activity, this is known as insertional inactivation.