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Question #47702
The Question
A researcher attempts to clone a gene into a plasmid vector using the restriction enzyme EcoRI. They design PCR primers with the EcoRI recognition sequence (5'-GAATTC-3') placed exactly at the extreme 5' terminus, with no additional upstream nucleotides. After successful PCR amplification using Taq polymerase, the amplicons are treated with EcoRI and mixed with an EcoRI-digested vector and DNA ligase. However, no recombinant plasmids are formed. What is the most likely molecular reason for this failure?
Options
- A.
EcoRI requires extra flanking nucleotides upstream of its recognition site for stable binding and cleavage, resulting in uncut amplicons.
- B.
Taq polymerase adds a template-independent 3' adenine overhang that permanently destroys the EcoRI cohesive ends.
- C.
The EcoRI recognition sequence at the extreme 5' end denatured during the extension phase, preventing proper primer annealing.
- D.
DNA ligase requires blunt ends for cloning PCR products, whereas EcoRI generates incompatible sticky ends.
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Correct Answer
Step-by-Step Solution
Explanation
How the Examiner Thinks
Evolutionary_Exception
Tackle problems where standard rules evolve or change based on specific environmental constraints.
Concepts Behind This Question
Flocs_Formation|Agrobacterium_Tumefaciens_Mechanism|Microbes_in_Human_Welfare
Why This Question Is Tricky
Common Mistakes
Conceptual Pivot: Students learn from NCERT that restriction sites can be added to the 5' end of primers for cloning.
However, the 'Plus' depth requires understanding the physical constraints of the enzyme: it needs a 'landing pad' of flanking bases to grip the DNA.
A common misconception is confusing the interference of Taq's 3' A-overhangs (relevant for TA cloning) with the actual failure of the restriction enzyme to cleave terminal sequences.
Quick Facts About This Question
Question Information
| Subject | Biology |
| Chapter | Microbes in Human Welfare - Class 12 |
| Concept | Flocs Formation |
| Difficulty | 8 |
| Cognitive Load | 5 |
| Target Solve Time | 120 sec |
| Access | Free |