Non-Perturbative Tracking of Processive DNA Synthesis with Single-Molecule Fluorescence

We have demonstrated recently that double-stranded DNA labeled with a periodic series of fluorescent dyes can be used to track a single helicase. Here we describe how this technique can be modified to follow DNA synthesis. By means of a stepwise loss of fluorescence during strand displacement, we monitor processive motion of a single $\phi 29$ DNA polymerase without labeling or altering the enzyme or the template strand, and without applying any force. We observe a wide range of speeds, with the highest exceeding by several times that observed in other single-molecule experiments. Because this method enables repeated observations of the same polymerase traversing identical segments of DNA, it should prove useful for studying sequence-specific effects in DNA replication and transcription.