Electron microscopy of the melting of sequenced DNA.

Abstract

AbstractDifferential melting curves (DMCs) of DNAs pA03 and pBR322 in solutions of different ionic strength (0.02 and 0.2M Na+) were obtained. A previously developed procedure of glyxal fixation of partially denatured DNA molecules at temperatures within the melting range was used to construct electron‐microscopic melting maps for pBR322 and pAO3 plasmid DNA and for the replicative form of bacteriophage ϕX174 DNA, allowing the melting of these DNA molecules to be followed in solutions of low (0.1 × SSC) and high (1 × SSC) ionic strength. In spite of the fact that the melting was at nonequilibrium at the low ionic strength, the melting maps for the two kinds of solutions practically coincided. Experimental data are compared with theoretical calculations based on the Fixman‐Freire algorithm. The conclusion is that the melting pattern of these DNAs is, on the whole, correctly described by the theory, although there are appreciable differences between the theoretical and experimental differential melting curves. We have also determined the relation between the melting temperature of a region and its GC content, with allowances made for the boundary conditions of melting in 0.1 × SSC and 1 × SSC solutions, and have analyzed the theoretical shape of peaks of the DMCs.