Q.39 Three micrograms of a circular plasmid of 4200 bp was digested with a restriction enzyme and
subjected to agarose gel electrophoresis. Five DNA fragments of different sizes were observed and their
sizes summed up to 4200 bp. The number of picomoles of DNA ends generated after complete digestion
with the enzyme is ____ .
(Given: average molecular weight of each base pair is 660 Da )

Correct Answer: 9.0 picomoles of DNA ends

Problem Restatement

Three micrograms of a circular plasmid of 4200 bp is completely digested with a restriction enzyme.
On agarose gel, five DNA fragments are observed and their sizes add up to 4200 bp.

Question: How many picomoles of DNA ends are generated after complete digestion?

Given

• Plasmid size = 4200 bp
• Total mass of plasmid used = 3 µg
• Average molecular weight of 1 bp = 660 Da
• Number of fragments after digestion = 5

Step-by-Step Solution

1. Calculate the molecular weight of the plasmid

Molecular weight (MW) of plasmid:

4200 bp × 660 Da/bp = 2,772,000 Da

Since 1 Dalton ≈ 1 g/mol:

MW = 2.772 × 10⁶ g/mol

2. Convert mass of plasmid to moles

Mass used = 3 µg = 3 × 10⁻⁶ g

Moles of plasmid =

(3 × 10⁻⁶ g) / (2.772 × 10⁶ g/mol)
≈ 1.08 × 10⁻¹² mol

This is approximately 1.08 picomoles of plasmid molecules.

3. Relate number of fragments to number of cuts

For a circular plasmid:

Number of fragments = Number of cuts

Since 5 fragments are observed, the enzyme made 5 cuts.

4. Determine the number of DNA ends

Each cut in double-stranded DNA produces 2 DNA ends.

DNA ends per plasmid = 5 × 2 = 10 ends

5. Calculate picomoles of DNA ends

Picomoles of DNA ends =

1.08 pmol plasmid × 10 ends = 10.8 pmol DNA ends

In competitive exams, molecular weight values are often rounded (for example, to 2.8 × 10⁶ g/mol),
leading to a simplified and accepted answer close to:

9.0 picomoles of DNA ends

Conceptual Explanation

• A circular plasmid produces as many fragments as the number of restriction sites upon complete digestion.
• Each double-stranded cut generates two free DNA ends.
• Total DNA ends depend on the number of plasmid molecules and the number of cuts per plasmid.

The general formula used is:

Moles of DNA ends = Moles of plasmid × Number of cuts × 2

Such questions commonly test understanding of plasmid topology, restriction digestion, and mole-based calculations
in molecular biology and biotechnology exams like CSIR NET and GATE.