. Define Insertional inactivation
Best Answer
Explanation-
- 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.
Final Answer-
In plasmid pBR322 and pUC8, when the gene gets inactivated due to restriction endonuclease activity, this is known as insertional inactivation.
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