65. A linear double stranded DNA of length 8 kbp has three restriction sites.
Each of these can either be a BamHI or a HaeIII site.
The DNA was digested completely with both enzymes. The products were purified
and subjected to an end-filling reaction using the Klenow fragment and
[α-³²P]-dCTP.
The products of the end-filling reaction were purified, resolved by
electrophoresis, stained with ethidium bromide (EtBr) and then subjected to
autoradiography. The corresponding images are shown below.

Introduction (SEO-Optimized)

Restriction mapping using BamHI and HaeIII is a classic molecular biology MCQ topic that tests understanding of restriction enzymes, DNA end structures, radioactive end-labeling, and interpretation of autoradiographs. In this article, we solve the given MCQ step by step, explain the logic behind autoradiographic signal intensities, and analyze all four options to identify the correct restriction map of an 8 kbp linear DNA.

Understanding the Question

• Linear double-stranded DNA = 8 kbp

• Three restriction sites total

• Each site is either BamHI or HaeIII

• DNA is completely digested with both enzymes

• Fragments are subjected to end-filling using Klenow fragment + [α-³²P]-dCTP

• Products analyzed by:

  • EtBr-stained gel → shows fragment sizes

  • Autoradiograph → shows radioactively labeled ends

Key Concepts Needed

1. Nature of DNA Ends

• BamHI

  • Produces 5′ sticky ends

  • These recessed 3′ ends are filled in by Klenow

  • Hence radioactive dCTP is incorporated

• HaeIII

  • Produces blunt ends

  • No recessed ends → no radioactive labeling

🔑 Conclusion:
Only fragments containing BamHI ends appear on the autoradiograph.

2. Autoradiograph Signal Intensity

• Signal intensity ∝ number of BamHI ends present in a fragment

• Fragment types:

  • Terminal fragment with 1 BamHI site → lower signal

  • Internal fragment flanked by 2 BamHI sites → higher signal

Interpreting the Given Data

From EtBr-stained gel

Fragments observed:

• 2 kb

• 2 kb

• 3 kb

• 1 kb
  (Total = 8 kb ✔)

From Autoradiograph

• 2 kb band → intensity 300

• 3 kb band → intensity 900

👉 Ratio of intensities = 1 : 3

What Does the 1:3 Ratio Mean?

• 300 units → fragment has one BamHI end

• 900 units → fragment has three times more labeling

• This is consistent with:

  • One fragment having one BamHI site

  • Another fragment being adjacent to two BamHI sites (internal fragment)

Therefore:

• Two BamHI sites

• One HaeIII site

Option-Wise Explanation

Option (A) ❌ Incorrect

• Places two BamHI sites adjacent

• Produces fragment labeling patterns inconsistent with the 1:3 autoradiograph ratio

• Fragment intensities do not match experimental data

Option (B) ✅ Correct Answer

• Restriction order:

  BamHI —— BamHI —— HaeIII


• Fragment sizes:

  • 2 kb | 2 kb | 3 kb | 1 kb

• Labeling logic:

  • One fragment has 1 BamHI end → low signal (300)

  • One internal fragment flanked by BamHI sites → high signal (900)

✔ Perfectly matches EtBr gel + autoradiograph

Option (C) ❌ Incorrect

• BamHI sites placed at DNA ends

• Would produce equal or symmetric labeling

• Does not explain 1:3 intensity ratio

Option (D) ❌ Incorrect

• Contains two HaeIII sites

• Too few BamHI ends → autoradiograph signal would be weak or absent

• Contradicts observed strong labeling

Final Answer

✅ Correct Option: (B)

Summary Table

Exam Tip

In restriction mapping MCQs:

• Autoradiography = labeled ends

• Sticky ends + Klenow = radioactive incorporation

• Blunt ends = no signal

• Always correlate signal intensity with number of labeled ends