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Nucleic Acids Research Advance Access published online on January 20, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp006
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Real-time single-molecule observation of rolling-circle DNA replication

Nathan A. Tanner1, Joseph J. Loparo1, Samir M. Hamdan1, Slobodan Jergic2, Nicholas E. Dixon2 and Antoine M. van Oijen1,*

1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA and 2School of Chemistry, University of Wollongong, Wollongong, NSW 2522, Australia

*To whom correspondence should be addressed. Tel: +1 617 432 5586; Fax: +1 617 738 0516; Email: antoine_van_oijen{at}hms.harvard.edu

Received December 9, 2008. Revised December 31, 2008. Accepted January 5, 2009.

We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.


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