Visualization of alkali-denatured supercoiled plasmid DNA by atomic force microscopy

Abstract

To study the alkali denaturation of supercoiled DNA, plasmid pBR322 was treated with gradient concentrations of NaOH solution. The results of gel electrophoresis showed that the alkali denaturation of the supercoiled DNA occurred in a narrow range of pH value (12.88–12.90). The alkali-denatured supercoiled DNA ran, as a sharp band, faster than the supercoiled DNA. The supercoiled plasmid DNA of pBR322, pACYC184 and pJGX15A were denatured by NaOH, and then visualized by atomic force microscopy. Compared with the supercoiled DNA, the atomic force microscopy images of the alkali-denatured supercoiled DNA showed rough surface with many kinks, bulges on double strands with inhomogeneous diameters. The apparent contour lengths of the denatured DNA were shortened by 16%, 16% and 50% for pBR322, pACYC184 and pJGX15A, respectively. All evidence suggested that the alkali-denatured supercoiled DNA had a stable conformation with unregistered, topologically constrained double strands and intrastrand secondary structure.