A simple electrophoretic method for the determination of superhelix density of closed circular DNAs and for observation of their superhelix density heterogeneity
✍ Scribed by Romilio T. Espejo; Jacob Lebowitz
- Publisher
- Elsevier Science
- Year
- 1976
- Tongue
- English
- Weight
- 590 KB
- Volume
- 72
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A simple method for directly dete~in~ng the superhelix density of closed circular DNAs by ethidium bromide titration using agarose ge1 electrophoresis in tubes containing increasing concentrations of the dye has been deveIoped. Since the positively charged dye migrates toward the cathode in a direction opposite to that of the DNA, ethidium bromide need not be present in the buffer chambers if electrophoresis is stopped before the dye boundary meets the DNA band. In this way, a constant concentration of ethidium bromide is present throughout the path of the DNA band. Consequently, by avoiding the presence of dye in the buffer chambers, many gels containing different concentrations of et~idium bromide can be run at the same time in a conventions gel-tube electrophoresis apparatus. A dye titration can be performed in less than a day by using this method. At several dye concentrations, the closed circular DNA is resolved into distinct bands which probably reflect intrinsic heterogeneity in superhelical density.