Q.46 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:
(A) 2800, 700 and 500 (B) 2800, 700 and 800
(C) 2500, 700 and 800 (D) 2500, 1200 and 300
Given Information
- Insert DNA size = 1.2 kb (1200 bp)
- Vector backbone size = 2.8 kb (2800 bp)
- Restriction sites on vector:
BamHI and EcoRI are adjacent
XhoI site is 300 bp upstream of BamHI - Insert restriction map:
BamHI —— 500 bp —— XhoI —— 700 bp —— EcoRI (Total = 1200 bp) - Final digestion: EcoRI + XhoI
- Assumption: Complete digestion
Step 1: Structure of Recombinant Plasmid
After cloning:
- BamHI–EcoRI region of vector is replaced by 1.2 kb insert
- Vector still contains one XhoI site
- Insert contains one internal XhoI site
- Total two XhoI sites and one EcoRI site in recombinant plasmid
Step 2: Cutting Pattern with EcoRI + XhoI
- EcoRI cuts once
- XhoI cuts twice
- So the plasmid is cut into 3 fragments
Step 3: Fragment Size Calculations
Fragment 1 (Insert fragment: XhoI → EcoRI)
From insert map: XhoI to EcoRI = 700 bp
Fragment = 700 bp
Fragment 2 (Insert fragment: BamHI → XhoI)
From insert map: BamHI to XhoI = 500 bp
Fragment = 500 bp
Fragment 3 (Vector fragment: EcoRI → XhoI)
Vector backbone = 2800 bp
BamHI and EcoRI are adjacent → negligible distance
XhoI is 300 bp upstream of BamHI
So EcoRI → XhoI distance on vector = 300 bp
But remember: Insert replaces BamHI–EcoRI junction
Remaining vector length after removing BamHI–EcoRI = 2800 bp
So vector fragment includes: 2800 bp − 300 bp = 2500 bp
Fragment = 2500 bp
Final Fragments Seen on Gel
| Fragment | Size (bp) |
|---|---|
| Vector | 2500 bp |
| Insert | 700 bp |
| Insert | 500 bp |
Correct Answer
(C) 2500, 700 and 500 bp
Introduction
The restriction enzyme mapping EcoRI XhoI recombinant plasmid problem is a high-frequency question type in GATE Biotechnology and CSIR NET Life Sciences exams. These questions test your ability to interpret cloning strategies, restriction site positions, and predict DNA fragment sizes after enzymatic digestion. This article provides a clear and exam-ready solution to such a problem using logical mapping and base-pair calculations.
Concept Behind the Question
- Cloning replaces vector restriction sites with an insert
- Internal restriction sites in the insert remain intact
- Digestion with enzymes that cut multiple times produces multiple fragments
- Fragment size = distance between consecutive restriction sites
Step-by-Step Strategy
- Identify restriction sites on vector and insert
- Construct the recombinant plasmid map
- Count number of cuts → number of fragments
- Measure distances between enzyme sites
- Match fragment sizes with given options
Why Other Options Are Incorrect
| ❌ Option | Fragments | Error |
|---|---|---|
| (A) | 2800, 700 and 500 | Treats vector as intact Ignores internal XhoI digestion |
| (B) | 2800, 700 and 800 | Incorrect insert fragmentation Insert internal XhoI already splits it into 500 + 700 |
| (D) | 2500, 1200 and 300 | Assumes insert remains uncut Ignores internal XhoI site |
Exam Tip
Always check for internal restriction sites in the insert.
These are the most common traps in restriction mapping questions.
Conclusion
By carefully mapping EcoRI and XhoI sites on both the vector and insert, the digestion of the recombinant plasmid produces three DNA fragments: 2500 bp, 700 bp, and 500 bp. Mastering this approach ensures accuracy in molecular biology questions involving cloning and restriction analysis.
