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Ethidium binding affinity of circular lambda deoxyribonucleic acid determined fluorometrically.

D M Hinton, V C Bode

    The Journal of Biological Chemistry
    |February 10, 1975
    PubMed
    Summary
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    This study quantifies differences in ethidium binding between supercoiled and linear lambda DNA using fluorescence measurements. Salt concentration influences DNA supercoiling, affecting dye intercalation and helix geometry.

    Area of Science:

    • Molecular Biology
    • Biophysics
    • Biochemistry

    Background:

    • Ethidium bromide is a fluorescent intercalating agent used to study DNA structure.
    • Supercoiled DNA exhibits unique binding properties compared to linear DNA.
    • Ionic strength can influence DNA conformation and dye-DNA interactions.

    Purpose of the Study:

    • To determine ethidium-binding isotherms for circular and linear lambda DNA.
    • To investigate the effect of salt concentration on DNA-dye binding affinity.
    • To quantify supercoiling in circular DNA and its salt-dependent changes.

    Main Methods:

    • Fluorescence spectroscopy to measure ethidium-DNA binding.
    • Construction of ethidium-binding isotherms.
    • Experiments conducted in varying concentrations of NaCl.

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    Main Results:

    • Differences in ethidium binding affinity between supercoiled and linear DNA were observed and quantified at different NaCl concentrations.
    • Supercoiled lambda DNA initially bound ethidium more avidly than linear DNA, with affinity changing at higher dye concentrations.
    • Calculated supercoiling in circular lambda DNA increased with salt concentration, corresponding to a salt-dependent change in base pair rotation angle.

    Conclusions:

    • Salt concentration significantly impacts the binding of ethidium to supercoiled DNA.
    • The study provides a method to calculate supercoiling in circular DNA based on dye binding.
    • Results suggest a salt-dependent alteration in the helical twist of the DNA molecule.