Visualizing single-molecule DNA replication with fluorescence microscopy

Authors

Nathan Tanner, Harvard Medical School
Joseph J. Loparo, Harvard Medical School
Antoine M. van Oijen, Harvard Medical SchoolFollow

RIS ID

94079

Publication Details

Tanner, N. A., Loparo, J. J. & van Oijen, A. M. (2009). Visualizing single-molecule DNA replication with fluorescence microscopy. Journal of Visualized Experiments, 32 e1529-1-e1529-5.

Link to publisher version (URL)

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Abstract

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera. 2009 Journal of Visualized Experiments.