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Question 67 of 78

Q.(a) 'Insertional inactivation' is a method to detect recombinant DNA. Explain the method.

(OR)
(b) Explain how recombinant DNA technology is used to detect a disease even before any clinical symptom appears.
Telangana TsbieCBSE Class XII Board 2023Subjective· 2mImportance★★★★★
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Part (a): Insertional inactivation clones foreign DNA into a marker gene so the insertion destroys that gene's function, letting recombinants be selected as Amp-resistant/Tet-sensitive (or white lacZ colonies).

Part (b): PCR amplifies the suspect sequence and a labelled complementary DNA probe hybridises to it, revealing a mutation or low-level pathogen before symptoms appear.

Part (a)

Concept-first idea: After ligation we need to tell recombinant plasmids (carrying insert) from non-recombinant ones. Insertional inactivation does this by making a successful insertion destroy a detectable marker.

Method.

  1. Use a vector such as pBR322 carrying two antibiotic-resistance genes — e.g., ampicillin resistance (amp^R) and tetracycline resistance (tet^R).
  2. Insert foreign DNA at a restriction site located within one marker (say inside tet^R).
  3. The insertion interrupts and inactivates tet^R, so the recombinant cell can no longer make functional tetracycline-resistance protein, but amp^R stays intact.
  4. Select transformants on ampicillin (only cells with a plasmid survive), then replica-plate onto tetracycline:
    • Non-recombinants (empty vector) grow on both antibiotics.
    • Recombinants grow on ampicillin but not tetracycline.
  5. Colonies that are Amp-resistant, Tet-sensitive carry the recombinant DNA. …

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