Q.What is meant by 'insertional inactivation' of a marker gene? Explain how it is used to distinguish recombinant colonies from non-recombinant ones on an antibiotic-containing medium.
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Start your 14-day free trial to unlock the full solution →Insertional inactivation relies on positioning a vector's unique restriction (cloning) site inside one of its selectable marker genes -- for example, a BamHI site located within the tetR (tetracycline-resistance) gene of pBR322. If a foreign DNA fragment is inserted at that site, it physically interrupts the coding sequence of the marker gene, destroying its ability to produce a functional protein; the marker gene is said to be 'insertionally inactivated'.
This has a directly observable consequence: a host cell that has taken up a true recombinant plasmid (vector plus insert at that site) will have lost the function of that one marker -- for example, losing tetracycline resistance -- while any other marker gene on the vector that was left undisturbed (such as ampR) continues to function normally, so the cell remains resistant to ampicillin. A host cell that took up only the original, unmodified, self-ligated vector (with no insert at all) still has both marker genes intact, and so remains resistant to both antibiotics. …
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