Which of the following plasmid vectors can be used for cloning of a gene, with restriction enzymes BamHI and EcoRI, and ampicillin-containing nutrient agar for selection?
[Ori and $\text{Amp}^R$ represent origin of replication and gene for ampicillin resistance, respectively.]
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Always ensure cloning restriction sites are located outside key genetic elements (like Ori) and outside your target selection marker to avoid inactivation.
This is a core principle in designing recombinant DNA experiments.
Step 1: Understanding the Question:
We need to identify the correct plasmid vector configuration that allows cloning of a gene using the restriction enzymes BamHI and EcoRI, while maintaining the ability to select the transformants on ampicillin-containing agar. Step 2: Key Formula or Approach:
To successfully use a plasmid vector for cloning and selection:
1. The origin of replication (Ori) must remain fully functional so the plasmid can replicate inside the host.
2. The ampicillin resistance gene ($\text{Amp}^R$) must remain functional so the host cells can grow on ampicillin selection plates.
3. The restriction sites used for cloning (BamHI and EcoRI) must NOT lie inside either the Ori or the $\text{Amp}^R$ gene. If they do, digesting the plasmid with these enzymes and inserting a foreign gene will cause insertional inactivation (disruption) of these essential features. Step 3: Detailed Explanation:
Let's analyze the positions of the restriction sites in each option:
- Option (a): Both EcoRI and BamHI are located in a region external to both the $\text{Amp}^R$ gene and the Ori. Cloning a gene into these sites will leave both $\text{Amp}^R$ and Ori completely intact and functional, allowing easy selection on ampicillin agar.
- Option (b): The BamHI site is located directly inside the $\text{Amp}^R$ gene. If we clone using BamHI, the ampicillin resistance gene will be disrupted (insertional inactivation), and the transformed cells will not survive on ampicillin-containing agar.
- Option (c) and (d): The BamHI site is located inside the Ori. Disruption of Ori will prevent the plasmid from replicating altogether, rendering it useless as a cloning vector.
- Therefore, only the configuration in (a) is suitable. Step 4: Final Answer:
Thus, plasmid (a) is the only one that satisfies all conditions, making option (A) the correct choice.
