Question

Which of the following cannot be used for confirmation of positive clones?

• A. Colony hybridization
• B. Colony PCR
• C. Growing transformants on selection media
• D. Growing transformants under low temperature

Hint:

Confirmation techniques must target molecular differences, such as DNA sequence, fragment length, or specific insertional marker disruption.

Answer 1

The Correct Option is D

Step 1: Understanding the Concept:
Confirming positive clones requires verifying that host cells carry the recombinant plasmid containing the specific target insert.
This verification relies on methods that detect the sequence, size, or expression of the foreign DNA insert.

Step 2: Detailed Explanation:
Colony hybridization uses a labeled nucleic acid probe complementary to the target insert to confirm its presence.
Colony PCR utilizes primers flanking the insertion site to directly amplify the cloned insert from a bacterial colony.
Growing transformants on selection media (such as blue-white screening or dual-antibiotic media) verifies insertional inactivation or marker expression.
Each of these three techniques directly assesses the genetic makeup of the plasmid in the transformed cell.
However, growing transformants under low temperature is a general physical condition that only alters growth rate and cell metabolism.
It cannot distinguish between empty vectors and recombinant plasmids containing the desired gene.
Therefore, temperature modification cannot serve as a method for confirming positive clones.

Step 3: Final Answer:
Hence, the correct option is (D).