Q.45 A 7 kb DNA molecule of a specific sequence has two EcoRI and one PvuII restriction endonuclease
sites. The restriction sites are shown below. The DNA was completely digested with both EcoRI and
PvuII. The digestion product was purified and added to an appropriately buffered reaction mixture at
37 °C, which contained the Klenow fragment of DNA polymerase I and α–32P dNTPs. After one hour,
the DNA in the reaction product was purified and analyzed by electrophoresis. The bands were
visualized by both ethidium bromide (EtBr) staining and autoradiography. The result is shown below.
Which one of the following restriction maps is in agreement with the above result?
Key Concepts
- EcoRI generates 5′ sticky ends.
- PvuII generates blunt ends.
- Klenow fragment fills in sticky ends only.
- Autoradiography detects fragments that incorporated radioactive dNTPs.
- Blunt-ended fragments remain unlabeled.
Interpretation of EtBr-Stained Gel
The EtBr-stained gel shows all DNA fragments after digestion.
The observed bands are:
- 3.0 kb
- 2.0 kb
- 1.5 kb
- 0.5 kb
Total size = 3.0 + 2.0 + 1.5 + 0.5 = 7.0 kb, confirming complete digestion.
Interpretation of Autoradiogram
The autoradiogram shows only radioactively labeled fragments.
Observed bands are:
- 3.0 kb
- 2.0 kb
- 0.5 kb
The 1.5 kb fragment is missing, indicating that it has
blunt ends on both sides and was not labeled.
Fragment End Analysis
| Fragment Size | EtBr Staining | Autoradiogram | End Type |
|---|---|---|---|
| 3.0 kb | Yes | Yes | EcoRI |
| 2.0 kb | Yes | Yes | EcoRI |
| 0.5 kb | Yes | Yes | EcoRI |
| 1.5 kb | Yes | No | PvuII–PvuII |
Final Answer
The restriction map that matches both the EtBr-stained gel and the autoradiogram
is Option (D).
Conclusion
Only fragments with EcoRI-generated sticky ends incorporate radioactive nucleotides
during the Klenow fill-in reaction. The absence of the 1.5 kb band in the autoradiogram
confirms that it is flanked by blunt PvuII sites. Among all options, only Option (D)
satisfies the fragment sizes, labeling pattern, and linear DNA requirement.
