35. Given below are the maps of a 1200 base pairs (bp) long DNA insert and a 3000 bp expression
vector. The BamHI(𝐵) and HindIII ( 𝐻 ) restriction sites and DNA length between them are
indicated in base pairs.
The insert is cloned into the vector at the BamHI site and the desired orientation is shown by the
arrow. After cloning, the orientation of the insert in the recombinant plasmid is tested by
complete HindIII digestion followed by agarose gel electrophoresis. Which one of the following
band patterns reveals the correct orientation of the insert in the construct?

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Introduction

Determining the orientation of a DNA insert in a recombinant plasmid is a common and important application of restriction enzyme mapping in molecular cloning. In this problem, a 1200 bp DNA insert is cloned into a 3000 bp expression vector at the BamHI site, and the orientation is tested using complete HindIII digestion, followed by agarose gel electrophoresis.

Let’s decode the maps, predict fragment sizes, and identify the correct gel band pattern.

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Given Information

Insert Map (1200 bp)

• BamHI (B) at both ends

• Two HindIII (H) sites inside the insert

• Distances (from 5′ → 3′ direction shown by arrow):

  • B → H = 300 bp

  • H → H = 200 bp

  • H → B = 700 bp

Vector Map (3000 bp)

• One BamHI site (cloning site)

• One HindIII site

• Distance between HindIII and BamHI = 300 bp

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Cloning Strategy

• Insert is ligated into vector at BamHI site

• Orientation is not random—desired orientation is shown by the arrow

• Orientation check is done by complete HindIII digestion

👉 Only HindIII sites matter after cloning.

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Step 1: Total HindIII Sites After Cloning

🔹 A circular plasmid cut at 3 sites produces 3 DNA fragments.

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Step 2: Predict Fragment Sizes (Correct Orientation)

Following the correct orientation shown in the diagram:

1. Vector HindIII → First Insert HindIII
   = 300 bp (vector) + 300 bp (insert)
   = 600 bp

2. Between Insert HindIII sites
   = 200 bp

3. Remaining DNA
   = Total plasmid size − (600 + 200)
   = (3000 + 1200) − 800
   = 3400 bp

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Expected Bands After HindIII Digestion

✔ 3400 bp
✔ 600 bp
✔ 200 bp

(200 bp band may appear faint or near the bottom of the gel)

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Option-Wise Analysis

Option A

• Shows ~3000 bp and ~500 bp fragments

• ❌ Missing the largest (~3400 bp) fragment
  Incorrect

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Option B

• Shows ~3000 bp, ~1000 bp, and ~250 bp

• ❌ Fragment sizes do not match calculated values
  Incorrect

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Option C

• Shows ~3400 bp, ~600 bp, and ~200 bp

• ✔ Exactly matches expected HindIII digestion pattern
  ✅ Correct Answer

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Option D

• Shows ~3000 bp and ~1000 bp fragments

• ❌ Only two bands; three HindIII sites must give three fragments
  Incorrect

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Final Answer

✅ Correct Option: (C)

This band pattern correctly reflects the expected HindIII digestion fragments (3400 bp, 600 bp, and 200 bp) for the desired orientation of the insert.

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Key Exam Takeaways

• Number of fragments = number of restriction sites (for circular DNA)

• Orientation analysis relies on relative distances between restriction sites

• Always:

  1. Count restriction sites

  2. Calculate fragment sizes

  3. Match with gel patterns