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Question #47716
The Question
A researcher successfully ligates a 25 kb eukaryotic genomic DNA fragment into the BamHI site of the pBR322 cloning vector. After transforming the mixture into E. coli, recombinants are correctly identified via insertional inactivation (growth on Ampicillin, no growth on Tetracycline). The researcher then isolates the plasmid DNA and attempts to purify the recombinant plasmid using standard agarose gel electrophoresis and elution. Despite successful biological selection, the elution yields no distinct functional recombinant plasmid band. Which of the following best explains this technical failure?
Options
- A.
The large eukaryotic insert lacks a bacterial origin of replication, preventing the recombinant plasmid from multiplying inside the E. coli host.
- B.
The massive size of the recombinant plasmid exceeds the molecular sieving limits of the agarose matrix, causing conformational entanglement and preventing distinct band resolution.
- C.
The recombinant plasmid loses its uniform negative charge due to the large eukaryotic insert, causing it to migrate anomalously towards the cathode.
- D.
Insertional inactivation of the tet^R gene eliminates the specific restriction sites required to physically cut and elute the DNA from the agarose gel.
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Correct Answer
Step-by-Step Solution
Explanation
How the Examiner Thinks
Regulatory_Failure
Understand how NOT, EXCEPT, and logical constraints are used to manipulate exam results.
Concepts Behind This Question
Repressor Protein|Operator Site|High-Copy-Number Plasmid
Why This Question Is Tricky
Common Mistakes
Conceptual Pivot: Students often assume that if biological selection (antibiotic screening) is successful, downstream physical recovery (elution) is guaranteed.
They fail to integrate the physical limitations of the 'sieving effect' in gel electrophoresis.
The trap lies in distinguishing between biological viability (plasmid replication in the host) and physical extractability (migration of a >20kb plasmid through an agarose matrix).
Quick Facts About This Question
Question Information
| Subject | Biology |
| Chapter | Biotechnology : Principles and Processes - Class 12 |
| Concept | Plasmid vs Chromosomal DNA |
| Difficulty | 8 |
| Cognitive Load | 5 |
| Target Solve Time | 120 sec |
| Access | Free |