Biological applications of the atomic force microscope: Measuring the forces that hold DNA together, and, Molecular level, direct, real time observation of the interactions of RecA protein with double stranded DNA

Employing the Atomic Force Microscope (AFM), two biological systems are examined under biological conditions. The force holding together single base pairs of DNA is probed. Measuring the force required to rupture a perfect double helix, a double helix with a single mismatch, and a double helix with a double mismatch, arranged in a microarray, the need for precise determination of the AFM tip spring constant is precluded. The large volume of data collected required the implementation of cluster analysis, used for the first time in single molecule force spectroscopy. The rupture force of the double helix ranged from 10 to 80 pN depending on the length of the double helix. Importantly, we found that the force contribution from both AT and GC base pairs is the same.; RecA protein's interaction with double stranded DNA is studied using time resolved AFM. The RecA/DNA nucleofilament is formed under physiological conditions and imaged. Several different species are formed, and have a pitch of 10 nm. The assembly of the RecA/DNA complex is monitored by examining contour length change over time. The assembly pathway occurred via a slow nucleation step, followed by a rapid growth phase. The lag time was 25 minutes, and the growth time was 17 minutes. The maximum growth speed was 92 RecA per minute. The disassembly of the complex is monitored in real time under buffer at pH 7.4 and 5.6, at a time resolution on the order of 2 minutes, and a spatial resolution better then 50 nm. The complexes disassemble from multiple discrete nodes in a non-random manner. At pH 7.4 the disassembly is rapid (up to 39 monomers per minute per node), and two to four times slower (as slow as 4.5 monomers per minute per node) at pH 5.6.; The design and implementation of a hot stage is described. The hot stage employs a simple, cost-effective design that allows a user to easily heat an AFM sample to 37 Celsius for experiments on biological systems. This is demonstrated by imaging DNA at ambient and physiological temperature.