Denaturing gradient gel electrophoresis (formamide with urea) has been used to study the melting of supercoiled DNA. A linegr gradient of denaturant concentration proportional to a 25°C linear increase of temperature (Teff) from the left to the righ edge of the gel was created perpendicular to DNA migration. The mobility of supercoiled DMA molecules was shown to drop to the level of relaxed molecules a long way (5-30°C) before linear DNA began to melt. The futher increase of Teff, including the roelting range for linear molecules, caused no appreciable changes in the mobility of relaxed molecules. The transition curves are S-shaped for all the topoisoraers, 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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