43. Shown below is a plasmid vector (P) and an insert (Q). The insert was cloned into the BamHI site of
the vector. The recombinant plasmid was isolated and digested with BamHI or XhoI. The results from the
digestion experiments are shown in (R).
Which one of the following explains the digestion results shown in (R)?
(a) The insert did not ligate to the vector.
(b) One copy of the insert ligated to the vector.
(c) The insert ligated to the vector as two tandem copies.
(d) The insert ligated to the vector as two copies but not in tandem.
Here, a 5 kb plasmid (P) receives a 1 kb insert (Q) ligated at a BamHI site. The recombinant plasmid is digested with BamHI and XhoI, generating the patterns shown in (R).
To determine what happened during cloning, we analyze: expected fragment sizes, observed digestion bands, and logical consistency with cloning outcomes.
Step-by-Step Interpretation
Vector & Insert Details
- Vector size: Plasmid backbone = 5 kb
- Insert size: Insert = 1 kb (with BamHI at both ends, XhoI inside)
What the Gel Shows (R):
XhoI digest: → produces 1 kb + 6 kb bands
Total size = 7 kb (5 kb vector + 2 × 1 kb inserts)
This indicates two copies of the insert are present, increasing plasmid from 5 kb → 7 kb.
Why it Must Be Tandem Copies
- BamHI cuts outside inserts → but inserts have BamHI ends → both copies ligate head-to-tail
- XhoI sits inside each insert, only two sites total, giving:
- One 1 kb fragment (internal toothpick)
- One 6 kb fragment containing vector + remaining insert copy
- This matches R exactly
Option-by-Option Elimination
(a) The insert did not ligate to the vector
Incorrect
Predicted plasmid stays 5 kb. Digests would still show 5 kb, not 7 kb.
(b) One copy of the insert ligated to the vector
Incorrect
Plasmid size should be 6 kb (5 + 1). XhoI digest would generate two fragments: ~1 kb and ~5 kb. Not seen on gel.
(c) Two tandem insert copies
✔ Correct
Total plasmid becomes 7 kb. XhoI yields 1 kb + 6 kb bands. BamHI yields single 7 kb band. Perfect match to R.
(d) Two copies ligated, but not in tandem
Incorrect
Opposite orientation inserts add extra restriction sites. XhoI would cut multiple times → 3 bands, not 2. Pattern does not match R.
Key Takeaways
The digestion pattern conclusively demonstrates:
- The insert ligated twice
- Both copies oriented head-to-tail (tandem)
- Final Answer: (c)
