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Question #47753
The Question
A researcher designs PCR primers containing EcoRI recognition sites at their 5' ends to amplify a specific gene for cloning. During the PCR cycle, the annealing temperature is accidentally set significantly lower than the optimal Tm (melting temperature) dictated by the primers' GC content. Which of the following is the most likely downstream consequence during the recombinant DNA construction process?
Options
- A.
Non-specific primer binding will generate unintended amplicons flanked by EcoRI sites, creating competing fragments during digestion that sabotage specific ligation.
- B.
Taq polymerase will be unable to synthesize DNA because the low temperature irreversibly denatures the enzyme during the extension phase.
- C.
The EcoRI enzyme will fail to cleave the PCR products because the low annealing temperature induces methylation of the newly synthesized DNA.
- D.
The primers will fail to anneal entirely due to decreased hydrogen bonding stringency, resulting in the complete absence of any PCR product.
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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
Agrobacterium Tumefaciens Mechanism|Heat Shock Thermal Gradient|Gene Transfer
Why This Question Is Tricky
Common Mistakes
Conceptual Pivot: Students often confuse the effects of high vs. low annealing temperatures in PCR.
High temperatures prevent annealing (high stringency), while low temperatures allow mismatched, non-specific annealing (low stringency).
The question tests the synthesis of this PCR concept with downstream restriction digestion and ligation, highlighting how an upstream error (non-specific amplicons with engineered restriction sites) fundamentally alters the recombinant DNA outcome.
Quick Facts About This Question
Question Information
| Subject | Biology |
| Chapter | Biotechnology : Principles and Processes - Class 12 |
| Concept | Heat Shock Thermal Gradient |
| Difficulty | 8 |
| Cognitive Load | 5 |
| Target Solve Time | 120 sec |
| Access | Free |