Question

A 2 kb DNA fragment was cloned into BamHI and EcoRI sites located on a 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. 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

Hint:

To solve plasmid mapping problems, sum the sizes of the fragments in each option. The correct option must equal the total size of the recombinant plasmid (4000 bp). Then, calculate the distance between the two known vector sites to find the large fragment size.

Answer 1

The Correct Option is C

Step 1: Understanding the Concept:
This problem requires calculating the sizes of DNA fragments produced by restriction digestion of a circular recombinant plasmid.
To find the fragment sizes, we must map the positions of the restriction sites relative to each other on the circular plasmid map.
Key Formula or Approach:
The total size of the recombinant plasmid is the sum of the vector size and the insert size:
\[ \text{Total Size} = \text{Vector Size} + \text{Insert Size} \]
Using the given values:
\[ \text{Total Size} = 2800\text{ bp} + 1200\text{ bp} = 4000\text{ bp} \]
Let us set the EcoRI site as the reference point ($0\text{ bp}$) on this circular $4000\text{ bp}$ map.

Step 2: Detailed Explanation:
The cloning sites are adjacent, meaning the $1200\text{ bp}$ insert is located between the BamHI and EcoRI sites.
If the EcoRI site is at coordinate $0\text{ bp}$ (or $4000\text{ bp}$), the BamHI site is located adjacent to it.
Since the insert is $1200\text{ bp}$ long, it spans from coordinate $0\text{ bp}$ to $1200\text{ bp}$.
The remaining $2800\text{ bp}$ of the plasmid consists of the vector backbone, spanning from $1200\text{ bp}$ back to $4000\text{ bp}$.
The vector contains an XhoI site located $300\text{ bp}$ upstream of the BamHI site.
This position corresponds to coordinate:
\[ 4000\text{ bp} - 300\text{ bp} = 3700\text{ bp} \]
The recombinant plasmid is digested to completion with EcoRI and XhoI.
The digestion sites are:
One EcoRI site at coordinate $1200\text{ bp}$ (the insert-vector junction).
One vector XhoI site at coordinate $3700\text{ bp}$.
One internal XhoI site within the insert, located at an unknown position $x$ between $0\text{ bp}$ and $1200\text{ bp}$.
This digestion yields three fragments:
- Fragment 1 is between the EcoRI site ($1200\text{ bp}$) and the vector XhoI site ($3700\text{ bp}$):
\[ 3700\text{ bp} - 1200\text{ bp} = 2500\text{ bp} \]
This calculation confirms that one of the fragments must be $2500\text{ bp}$, which rules out options A and B.
- The remaining $1500\text{ bp}$ region of the plasmid is cleaved by the internal XhoI site at position $x$, producing two smaller fragments:
Fragment 2 (from $3700\text{ bp}$ through the $4000/0\text{ bp}$ junction to $x$): size = $300 + x$.
Fragment 3 (from $x$ to $1200\text{ bp}$): size = $1200 - x$.
The sum of these two fragments must be $1500\text{ bp}$ ($4000\text{ bp} - 2500\text{ bp} = 1500\text{ bp}$).
Let us test the remaining options:
- In Option C, the sizes are $700\text{ bp}$ and $800\text{ bp}$ (sum = $1500\text{ bp}$). If we set $300 + x = 700$, we find $x = 400\text{ bp}$, which is a valid position within the $1200\text{ bp}$ insert.
- In Option D, the sizes are $1200\text{ bp}$ and $300\text{ bp}$ (sum = $1500\text{ bp}$). Setting $300 + x = 300$ yields $x = 0$, meaning the internal XhoI site would overlap with the cloning site, which contradicts the problem.
Therefore, the correct fragment sizes are $2500\text{ bp}$, $700\text{ bp}$, and $800\text{ bp}$.

Step 3: Final Answer:
The DNA fragments visible on the gel correspond to 2500, 700, and 800 bp.