Figure 8.

DNA synthesis by immobilized complexes. Purified ternary complexes made with unlabeled streptavidin were immobilized in a reaction chamber on a PEG–biotin coated coverglass (see Materials and methods section). (A) The reaction chamber was filled with 1 μl of buffer C containing 5 mM MgCl2, 100 μM each of dATP, dCTP, dGTP and base-labeled Alexa Fluor-488-dUTP (Invitrogen). The chamber was sealed with a plastic coverslip and incubated in a humid jar at 54°C for 90 min. The chamber was rinsed with water to remove unincorporated nucleotides and the coverglass surface was imaged by TIRF microscopy (see Materials and methods section). The labeled complexes were seen waving back and forth under Brownian motion, while remaining tethered to the surface (Supplementary Movie M1). (B) Control reaction inhibiting polymerase activity by replacing Mg++ with 0.1 mM EDTA. (C) Zoomed-in view of a single DNA spot from Movie M1 showing movement of about 1 μm leftward occurring between frames 61 and 62. A pixel is marked for reference (x–y coordinate 386, 534). Exposure time was 80 ms and the pixel dimension 0.27 μ. Movie M1 was acquired in a replicate experiment as in (A).