Section 3: Genetics, Cellular and Molecular Biology
Recombinant DNA technology
Subtopic: Fundamentals of Recombinant DNA technology
Question Statement:
The schematic of a plasmid with a gap in one of the strands is shown below:
ATCGCGATG
TAGC
AC
3' OH 5' OH
Which of the following enzyme(s) is/are required to fill the gap and generate a
covalently closed circular plasmid?
[P] DNA ligase
[Q] Alkaline phosphatase
[RJ DNA polymerase
[S] Polynucleotide kinase
Options:
(A) P only
(B) P, R and S only
(C) P and R only
(D) P, Q and R only
Official Answer Key:
B
Step-by-Step Solution & Explanation
### **Official Solution & Concept Analysis (GATE BT 2020 Q.47)**
**1. Governing Concept**:
- **Topic**: Recombinant DNA Technology & DNA Gap Repair
- A circular plasmid containing a single-stranded gap in one of its strands:
- Repair requires **DNA Polymerase I** (which synthesizes DNA filling the gap using the opposite intact strand as template) and **DNA Ligase** (which seals the remaining phosphodiester nick with ATP/$\text{NAD}^+$).
- Transformation into a repair-proficient *E. coli* host (e.g., $recA^+$ or wild-type polA/ligA) repairs the gap in vivo.
- Matches Option (B).
**2. Option Analysis**:
- **Option (A) is INCORRECT**: Ligase alone cannot seal a multi-nucleotide gap without prior polymerase filling.
- **Option (B) is CORRECT**: Requires DNA polymerase to fill the gap followed by DNA ligase to seal the nick.
- **Option (C) is INCORRECT**: Exonuclease would widen the gap.
- **Option (D) is INCORRECT**: Kinase alone is insufficient.
**3. Final Answer**:
- **Correct Choice**: **B**
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