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Topoisomerase relieves supercoiling tension in DNA by cutting and rejoining strands.
Ref: NCERT Class 12 Biology Chapter 5: Molecular Basis of Inheritance DNA Replication - Semiconservative and Machinery
Ethidium bromide (EtBr) is an intercalating dye that inserts between stacked base pairs, causing DNA to unwind and lengthen. Supercoiled closed-circular DNA is topologically constrained and can accommodate limited intercalation compared to relaxed or linear DNA. Intercalation reduces superhelical density and increases molecular volume, decreasing buoyant density in CsCl gradient. Linear and nicked DNA bind more EtBr and become less dense than supercoiled. Consequently, supercoiled plasmid bands lower in gradient at higher density, while EtBr-bound linear DNA bands higher, and this decreases buoyant density of intercalated DNA overall.
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.
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