Introduction

This solved example demonstrates how to construct a
restriction map of a circular plasmid using single and
double digests with EcoRI and BamHI, along with functional information
about a SalI site disrupting the ampicillin resistance gene cassette.
Such restriction map circular plasmid EcoRI BamHI SalI ampicillin resistance gene
questions are common in GATE and CSIR NET Life Sciences.

Step 1: Information from Single Digests

EcoRI digest:

• Fragments: 3.2 kb and 3.0 kb
• Total = 6.2 kb → two EcoRI sites

BamHI digest:

• Fragments: 5.0 kb and 1.2 kb
• Total = 6.2 kb → two BamHI sites

For circular DNA, the number of fragments produced by a single enzyme
equals the number of cut sites. Therefore, the plasmid length is
6.2 kb with two EcoRI and
two BamHI sites.

Step 2: Constraints from Double Digest

Double digestion with EcoRI + BamHI yields four fragments:
2.6 kb, 2.4 kb, 0.8 kb, and 0.4 kb.

Their sum equals 6.2 kb, confirming consistency. With two EcoRI and two
BamHI sites on a circle, the maximum number of double-digest fragments
is four, corresponding to the four intervals between alternating sites.

Step 3: Matching Single and Double Digest Data

Each single-digest fragment must be the sum of two adjacent double-digest
segments.

Possible sums of adjacent segments:

• 2.6 + 2.4 = 5.0
• 2.6 + 0.4 = 3.0
• 2.4 + 0.8 = 3.2
• 0.8 + 0.4 = 1.2

Observed EcoRI fragments are 3.2 kb and
3.0 kb, corresponding to
2.4 + 0.8 and 2.6 + 0.4.

Observed BamHI fragments are 5.0 kb and
1.2 kb, corresponding to
2.6 + 2.4 and 0.8 + 0.4.

A consistent circular arrangement is:

EcoRI — 2.4 kb — BamHI — 0.8 kb — EcoRI — 0.4 kb — BamHI — 2.6 kb — (back to EcoRI)

Step 4: Position of the SalI Site

Digestion with SalI disrupts the ampicillin resistance gene, indicating
that the SalI site lies within the Amp^r cassette.
In the correct restriction map, SalI must be positioned inside the
ampicillin resistance region rather than outside it.

This reflects classic insertional inactivation, as seen
in plasmids like pBR322.

Interpreting and Rejecting Typical Options

The correct option must satisfy all of the following:

• Total plasmid size = 6.2 kb
• EcoRI fragments = 3.2 kb and 3.0 kb
• BamHI fragments = 5.0 kb and 1.2 kb
• Double digest fragments = 2.6, 2.4, 0.8, 0.4 kb
• SalI site located within the Amp^r cassette

Option D satisfies all these constraints and is therefore
the correct restriction map.

Key Exam Tips

• For circular DNA, number of fragments equals number of cut sites.
• Single-digest fragments reveal both site count and plasmid size.
• Double-digest fragments represent individual intervals between alternating sites.
• A restriction site within an antibiotic resistance cassette causes gene disruption.

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