Abstract
Denaturing gradient gel electrophoresis (formamide with urea) has been used to study the melting of supercoiled DNA. A linear gradient of denaturant concentration proportional to a 25 degrees C linear increase of temperature (Teff) from the left to the right edge of the gel was created perpendicular to DNA migration. The mobility of supercoiled DNA molecules was shown to drop to the level of relaxed molecules a long way (5-30 degrees C) before linear DNA began to melt. The further increase of Teff, including the melting range for linear molecules, caused no appreciable changes in the mobility of relaxed molecules. The transition curves are S-shaped for all the topoisomers, and an increase of superhelicity shifts the transition towards lower Teff values. The analysis of the results indicates that the observed relaxation of superhelical molecules is due to denatured region forming in them, their size increasing with the topoisomer number.
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