Q75. One mole of a circular bacterial plasmid was digested with a high–fidelity
restriction enzyme. The plasmid has five restriction sites for the enzyme
used. The number of moles of fragments released upon cleavage at all sites
is________.
The number of moles of fragments released is 5.
A circular bacterial plasmid with five distinct restriction sites for a high-fidelity enzyme produces exactly five linear fragments when fully digested at all sites, as each cut opens the circle into segments equal to the number of cuts. Since one mole of intact plasmids is digested completely, one mole of each unique fragment is generated, totaling five moles of fragments. High-fidelity ensures precise cleavage without star activity or incomplete cuts.
Key Concept
Restriction enzymes cleave DNA at specific recognition sites. For circular DNA like plasmids, n sites yield n fragments, unlike linear DNA where n sites yield n+1 fragments.
Detailed Solution
Visualize a circular plasmid as a loop. Five cuts at distinct sites divide it into five arcs, each becoming a separate linear fragment post-digestion. Starting with 1 mole of plasmids (each ~3-10 kb typically), complete digestion releases 5 moles of fragments (1 mole per fragment type). No molar excess occurs, as fragments are equimolar from stoichiometric cleavage.
Common Misconceptions
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Linear DNA confusion: Might suggest 6 moles (5+1), but plasmids are circular.
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Incomplete digestion: High-fidelity enzyme and “cleavage at all sites” imply full cutting.
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Supercoiled forms: Digestion linearizes all, yielding only fragment moles.
In molecular biology, understanding circular bacterial plasmid digestion by restriction enzymes is crucial for CSIR NET Life Sciences aspirants. This guide solves: “One mole of a circular bacterial plasmid was digested with a high-fidelity restriction enzyme. The plasmid has five restriction sites for the enzyme used. The number of moles of fragments released upon cleavage at all sites is________.”
Why 5 Moles?
A circular bacterial plasmid acts like a closed loop. A high-fidelity restriction enzyme cuts precisely at each of the five sites, breaking the circle into five linear DNA fragments. From 1 mole of plasmids, you get 1 mole of each fragment, totaling 5 moles—no more, no less.
Step-by-Step Reasoning
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Confirm circular topology: Unlike linear DNA (n sites → n+1 fragments), circles give n fragments for n cuts.
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High-fidelity ensures complete, specific cleavage without off-target cuts.
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Stoichiometry: 1 mole plasmids × 5 fragments/molecule = 5 moles fragments.
Exam Options Explained
Though fill-in-the-blank, typical MCQ traps:
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4 moles: Underestimates cuts.
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5 moles: Correct for circle.
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6 moles: Linear DNA error.
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1 mole: Thinks single linear product.
Applications in Biotech
This principle underpins plasmid mapping, cloning, and recombinant DNA tech. Visualize on agarose gels: 5 bands if sizes differ.
