Figure 4.

KF DNA-polymerization assay. (A) Schematic. Initially the predominantly ssDNA substrate has a FIW close to that of the diffraction limit. As the DNA is converted to double stranded during polymerization, the mean persistence length increases, broadening the FIW until polymerization is complete. (B) Predicted FIW during polymerization. A linear fit to the region from 200 bp onward extracts an approximate conversion of 25 bases/nm. (C) Time traces from six polymerizing molecules. Dashed lines indicate the drop in fluorescence associated with the completion of polymerization, because of the conversion from single- to double-stranded of the DNA in the vicinity of the fluorophore. The straight lines are linear least squares fits to the actively polymerizing segments of the time traces, defined as from 50 s after the start of data acquisition until 20 s before the end of polymerization. The gradients of these fits can be used to extract a mean polymerization rate of 4.5 ± 0.7 bases/s. Molecules were selected to have not bleached before the end of the movie. In some (50%) of the time traces a small PIFE is evident before the completion of polymerization, which can be attributed to the proximity of KF to the fluorophore. To see this figure in color, go online.