Q.45 The figure below is a schematic of a linear double stranded DNA containing the indicated
restriction sites. The DNA was completely digested with PvuII and EcoRI. The products were
purified and added to an appropriately buffered reaction mixture containing dNTP mix, α32P
dATP, and Klenow fragment of E. coli DNA polymerase I. The Klenow reaction products were
analyzed by gel electrophoresis and autoradiography. Which of the following products depicts the
expected result?

Klenow Fragment End-Labeling after PvuII and EcoRI Digestion

Restriction digestion followed by end labeling using the Klenow fragment is a classic
molecular biology technique used to identify DNA fragments containing sticky ends.
This question is frequently asked in CSIR-NET, GATE, JAM, and MSc entrance exams.

Understanding the DNA Map

The given linear double-stranded DNA contains the following restriction sites:

• PvuII site at 2 kb from the left end
• EcoRI site 3 kb downstream of PvuII
• Total DNA length = 6 kb

Fragments after complete digestion:

1. 2 kb fragment (Left end → PvuII)
2. 3 kb fragment (PvuII → EcoRI)
3. 1 kb fragment (EcoRI → Right end)

Restriction Enzyme Properties

PvuII

• Recognition site: CAGCTG
• Produces blunt ends
• No recessed 3′ end → No Klenow labeling

EcoRI

• Recognition site: GAATTC
• Produces 5′ sticky ends
• Recessed 3′ ends → Klenow can label

Role of Klenow Fragment

The Klenow fragment of E. coli DNA polymerase I extends recessed 3′ ends.
When α-³²P dATP is present, radioactive labeling occurs only at sticky ends.

Which DNA Fragments Get Labeled?

Conclusion: Only the 3 kb and 1 kb fragments will appear on the autoradiograph.

Correct Answer

✅ Option (C)

Shows two radioactive bands at 3 kb and 1 kb.

Explanation of All Options

Option (A)

Displays incorrect or incomplete labeling pattern.
Does not clearly follow Klenow labeling logic.
Incorrect.

Option (B)

Shows labeling of the 2 kb fragment, which has blunt ends.
Klenow cannot label blunt ends.
Incorrect.

Option (C)

Shows labeling only at 3 kb and 1 kb fragments.
Matches EcoRI sticky-end labeling.
Correct.

Option (D)

Includes 2 kb fragment labeling, which is not possible.
Incorrect.

Exam Tip

Golden Rule:
Only DNA fragments with sticky ends and recessed 3′ ends are labeled by the Klenow fragment.

• Blunt ends → No labeling
• EcoRI → Labeling
• PvuII → No labeling

Final Conclusion

This is a high-yield molecular biology question integrating restriction digestion,
enzyme specificity, and autoradiography interpretation.

✔ Correct Answer: Option (C)