Visualizing Single-molecule DNA Replication with Fluorescence Microscopy.
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.
Tanner
Nathan A
NA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School.
Loparo
Joseph J
JJ
van Oijen
Antoine M
AM
D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D004261:DNA Replication; D008856:Microscopy, Fluorescence / Q000379:methods*; D013698:Templates, Genetic
D004247:DNA