![Q.64 The product of complete digestion of the plasmid shown below with EcoRI and HaeIII was purified and used as a template in a reaction containing Klenow fragment of DNA polymerase, dNTPs and [α-32P]-dATP in a suitable reaction buffer. The product thus obtained was purified and subjected to gel electrophoresis followed by autoradiography. EcoRI 5′–GAATTC–3′ 3′–CTTAAG–5′ HaeIII 5′–GGCC–3′ 3′–CCGG–5′ The number of bands that will appear on the X-ray film is ________](data:image/svg+xml;base64,PHN2ZyB2aWV3Qm94PSIwIDAgMTkyMCAxMDgwIiB3aWR0aD0iMTkyMCIgaGVpZ2h0PSIxMDgwIiB4bWxucz0iaHR0cDovL3d3dy53My5vcmcvMjAwMC9zdmciPjwvc3ZnPg==)
Q.64 The product of complete digestion of the plasmid shown below with EcoRI and HaeIII was purified and used as a template in a reaction containing Klenow fragment of DNA polymerase, dNTPs and [α-32P]-dATP in a suitable reaction buffer. The product thus obtained was purified and subjected to gel electrophoresis followed by autoradiography.
EcoRI
5′–GAATTC–3′
3′–CTTAAG–5′
HaeIII
5′–GGCC–3′
3′–CCGG–5′
The number of bands that will appear on the X-ray film is ________
EcoRI + HaeIII Digestion and Klenow Labeling: How Many Bands on Autoradiography?
- Klenow fragment of DNA polymerase
- dNTPs
- α-32P-dATP
Labeled product is run on gel → How many bands will appear on the autoradiogram?
Step 1 — Identify Restriction Sites
The map shows three cut sites:
- EcoRI – cuts once
- HaeIII – cuts twice
Fragment sizes produced:
- 3 kb fragment (HaeIII → EcoRI)
- 1 kb fragment (EcoRI → HaeIII)
- 5 kb fragment (HaeIII → HaeIII)
So digestion yields three physical fragments.
Step 2 — Which Fragments Will Be Radiolabeled?
Restriction End Behavior
| Enzyme | End Type | Fill-in Needed? | Incorporation of α-32P-dATP? |
|---|---|---|---|
| EcoRI | 5′ overhang | ✔ Yes | ✔ Yes (Klenow fills recessed 3′ ends) |
| HaeIII | Blunt | ❌ No | ❌ No incorporation |
Consequence
EcoRI produces 2 ends → both need filling → both pick up 32P-dATP
Ends created by HaeIII remain blunt → no label added
Step 3 — Which Fragments Carry Labeled Ends?
Because the single EcoRI site lies between two HaeIII sites, each EcoRI end belongs to a different fragment:
| Fragment | Ends Present | Labeled? |
|---|---|---|
| 3 kb | One EcoRI end + One HaeIII end | ✔ Yes |
| 1 kb | One EcoRI end + One HaeIII end | ✔ Yes |
| 5 kb | Two HaeIII ends | ❌ No |
Final Answer
Number of radioactive bands = 2
Only the 3 kb and 1 kb fragments incorporate radioactive dATP, so two bands appear on the X-ray film, even though three fragments exist on the gel.
Why Not Other Answers?
| Possible Answer | Why It’s Wrong |
|---|---|
| 1 band | Requires both labeled ends on same fragment — not the case |
| 3 bands | Only 2 fragments are labeled; the 5 kb remains cold |
| More than 3 | Only 3 fragments exist; no additional products |
Concept Takeaway
Autoradiography shows only radioactive DNA, not all fragments.
Because only EcoRI cut sites incorporate ³²P, the gel shows two labeled fragments.
Correct Answer: 2 bands
