Scanning force microscopy of circular and linear plasmid DNA spread on mica with a quaternary ammonium salt

Abstract

Scanning force microscopy (SFM) offers the potential for subnanometer resolution in the investigation of nucleic acids, proteins, and their complexes. SFM is not bound by the requirement of classical transmission electron microscopy (TEM) for contrast enhancement through shadow casting or negative staining. A primary challenge, however, has been the reproducible fixation of samples on an atomically flat surface such as mica. We have developed a method for the routine imaging by SFM of supercoiled, relaxed, and linearized plasmid DNA, immobilized on freshly cleaved mica through the spreading action of benzyldimethylalkylammonium chloride (BAC) at micromolar concentrations. A reproducibly high yield of well-spread, dispersed molecules is obtained and background contamination is minimal. The contour lengths of the relaxed and linearized molecules imaged in air agree well with the helical rise (3.4 A/bp) of B-DNA in solution. We have also introduced the use of quantitative image analysis of SFM images to determine apparent molecular width and height over the entire molecular path.