Question

The given picture was obtained from an agarose gel electrophoresis of a plasmid after digestion with restriction enzymes either X, Y or both X and Y.

Which one of the following diagrams correctly represents the position of the restriction enzyme sites (X, Y) on the 10,000 bp plasmid?

• A. A
• B. B
• C. C
• D. D

Hint:

For double digestions of circular DNA, check if the sum of all fragment lengths equals the total plasmid length.
Here, the bands are $3$ kb and $2$ kb, which sum to $5$ kb.
Since the plasmid is $10$ kb, there must be duplicate fragments ($2 \times 3$ kb + $2 \times 2$ kb = $10$ kb), meaning the restriction sites must alternate around the circle.

Answer 1

The Correct Option is A

Step 1 : Understanding the Question:
This question requires constructing a restriction map of a $10,000$ base-pair (bp) circular plasmid based on the fragment sizes obtained after single and double enzymatic digestions shown on an agarose gel.
Step 2 : Key Formulas and Approach:
The approach involves calculating the distances between restriction sites on a circular map of total size $10,000$ bp.
For a circular plasmid, the number of fragments produced by digestion equals the number of restriction sites present.
We will check each option to see which mapping matches the fragment lengths observed on the gel.
Step 3 : Detailed Explanation:

Analyzing Gel Bands:
Lane 1 (X alone): Yields a single band at $5$ kb ($5000$ bp). This means there are two X sites located exactly $5000$ bp apart, which yields two fragments of $5000$ bp running as a single band.

Lane 3 (Y alone): Also yields a single band at $5$ kb ($5000$ bp), indicating two Y sites located exactly $5000$ bp apart.

Lane 2 (X + Y double digest): Yields two bands at $3$ kb ($3000$ bp) and $2$ kb ($2000$ bp). This indicates that the four restriction sites (two X and two Y) partition the $10,000$ bp plasmid into alternating fragments of $3000$ bp and $2000$ bp (summing to $10,000$ bp).


Testing Option (A):
X sites are placed at $9235$ bp and $4235$ bp. The distance is $9235 - 4235 = 5000$ bp. This matches the single digest result.

Y sites are placed at $1235$ bp and $6235$ bp. The distance is $6235 - 1235 = 5000$ bp. This also matches the single digest result.

Arranging all four sites in sequential order along the circular plasmid: Y ($1235$ bp) $\rightarrow$ X ($4235$ bp) $\rightarrow$ Y ($6235$ bp) $\rightarrow$ X ($9235$ bp) $\rightarrow$ Y ($1235$ bp).

The distances between these adjacent sites are:
From Y($1235$) to X($4235$): $4235 - 1235 = 3000$ bp ($3$ kb)

From X($4235$) to Y($6235$): $6235 - 4235 = 2000$ bp ($2$ kb)

From Y($6235$) to X($9235$): $9235 - 6235 = 3000$ bp ($3$ kb)

From X($9235$) to Y($1235$) (across the origin): $(10000 - 9235) + 1235 = 2000$ bp ($2$ kb)



These calculations yield exactly two $3$ kb fragments and two $2$ kb fragments, which perfectly match the gel lanes.

Step 4 : Final Answer:
Thus, Option (A) is the only correct restriction map that explains the experimental gel data.