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Question #47710
The Question
A researcher digests a cloning vector with EcoRI and a passenger DNA with BamHI. They mix the purified fragments, add ATP and DNA ligase, and attempt to transform competent E. coli. Upon plating on a medium containing the antibiotic for the vector's intact selectable marker, zero colonies appear. Which of the following best explains the primary molecular reason for this failure?
Options
- A.
The non-complementary sticky ends fail to form transient hydrogen bonds, preventing DNA ligase from catalyzing phosphodiester bond formation and leaving the plasmid linear.
- B.
The mismatched sticky ends lead to insertional inactivation of the selectable marker, causing the transformed cells to die on the antibiotic medium.
- C.
DNA ligase requires blunt ends for efficient ligation; the 5' overhangs generated by EcoRI and BamHI competitively inhibit the enzyme's active site.
- D.
DNA ligase successfully joins the fragments, but the mismatched polarity creates a 3'-5' phosphodiester linkage that prevents plasmid replication.
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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
Restriction Endonuclease Nomenclature|Analogous Organ Structure|Restriction Enzyme Cleavage Physics
Why This Question Is Tricky
Common Mistakes
Conceptual Pivot: Students often memorize that 'DNA ligase joins DNA fragments' but forget the prerequisite: ligase requires the ends to be held together by transient hydrogen bonds between complementary sticky ends.
A common misconception (Option B) is jumping straight to 'insertional inactivation' whenever colonies fail to grow, ignoring the chronological sequence of recombinant DNA technology where successful ligation must precede transformation and expression.
Quick Facts About This Question
Question Information
| Subject | Biology |
| Chapter | Biotechnology : Principles and Processes - Class 12 |
| Concept | Restriction Enzyme Cleavage Physics |
| Difficulty | 8 |
| Cognitive Load | 5 |
| Target Solve Time | 120 sec |
| Access | Free |