Section 3: Genetics, Cellular and Molecular Biology
Recombinant DNA technology
Subtopic: Fundamentals of Recombinant DNA technology
Question Statement:
A 1.2 kb DNA fragment was cloned into BamHI and EcoRI sites located on a 2.8 kb cloning vector.
The BamHI and EcoRI sites are adjacent to each other on the vector backbone. The vector contains
an XhoI site located 300 bp upstream of the BamHI site. An internal XhoI site is present in the gene
sequence as shown in the figure. The resultant recombinant plasmid is digested with EcoRI and
XhoI and analyzed through 1% agarose gel electrophoresis. Assuming complete digestion with
EcoRI and XhoI, the DNA fragments (in base pairs) visible on the agarose gel will correspond to:
Options:
(A) 2800, 700 and 500
(B) 2800, 700 and 800
(C) 2500, 700 and 800
(D) 2500, 1200 and 300
Official Answer Key:
C
Step-by-Step Solution & Explanation
### **Official Solution & Step-by-Step Derivation (GATE BT 2016 Q.46)**
**1. Governing Concept & Question Classification**:
- **Section**: BT
- **Question Type**: **MCQ** (2 Marks)
- **Topic**: Recombinant DNA Technology (Restriction Mapping & Gel Electrophoresis Fragment Sizes)
**2. Detailed Step-by-Step Derivation**:
- Let us reconstruct the restriction map of the recombinant construct:
- **Vector Backbone**: Size = $2.8\text{ kb} = 2800\text{ bp}$.
- It contains a BamHI cloning site.
- An XhoI site is located $300\text{ bp}$ upstream of the BamHI site in the vector backbone.
- Thus, the distance from the EcoRI site in the vector to the BamHI site and flanking vector XhoI site is defined.
- **Cloned Insert**: Size = $1.2\text{ kb} = 1200\text{ bp}$.
- Cloned into the BamHI and EcoRI sites.
- An internal XhoI site is present in the insert $500\text{ bp}$ away from the BamHI junction, leaving the remaining segment to the EcoRI site as $1200 - 500 = 700\text{ bp}$.
- Now, digest the complete circular recombinant plasmid with **EcoRI and XhoI**:
1. Fragment 1: The segment between the insert internal XhoI site and the EcoRI site = **$700\text{ bp}$**.
2. Fragment 2: The segment between the vector XhoI site and the insert internal XhoI site spans the $300\text{ bp}$ vector region + $500\text{ bp}$ insert region = $300 + 500 = \mathbf{800\text{ bp}}$.
3. Fragment 3: The remaining vector backbone between the vector XhoI site and the EcoRI site = $2800 - 300 = \mathbf{2500\text{ bp}}$.
- Sum check: $700 + 800 + 2500 = 4000\text{ bp} = 4.0\text{ kb}$ (which equals $2.8\text{ kb vector} + 1.2\text{ kb insert}$).
- Therefore, the three distinct DNA fragments visible on the 1% agarose gel will correspond to:
$\mathbf{2500, 700, \text{ and } 800\text{ bp}}$
- **Option-by-Option Evaluation**:
- **Option (A) [INCORRECT]**: Lists 2800 and 500 bp, which assumes no cleavage at the vector XhoI site.
- **Option (B) [INCORRECT]**: Lists 2800 bp for the vector backbone.
- **Option (C) [CORRECT]**: 2500, 700, and 800 bp are the exact fragment sizes resulting from complete dual digestion with EcoRI and XhoI.
- **Option (D) [INCORRECT]**: Fails to add the contiguous internal segments.
**3. Final Answer / Verification Summary**:
- **Official GATE Answer Key**: **C**
- **Scoring Criteria**: +2.0 Marks (Negative marking: -0.67 marks).
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