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Nucleic Acids Research 2006 34(2):e15; doi:10.1093/nar/gnj016
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Published online 1 February 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

A general method for manipulating DNA sequences from any organism with optical tweezers

Derek N. Fuller, Gregory J. Gemmen, John Peter Rickgauer, Aurelie Dupont, Rachel Millin, Pierre Recouvreux and Douglas E. Smith*

Department of Physics, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0379, USA

*To whom correspondence should be addressed. Tel: +1 858 534 5241; Email: des{at}physics.ucsd.edu

Received July 16, 2005. Revised November 19, 2005. Accepted January 11, 2006.

Mechanical manipulation of single DNA molecules can provide novel information about DNA properties and protein–DNA interactions. Here we describe and characterize a useful method for manipulating desired DNA sequences from any organism with optical tweezers. Molecules are produced from either genomic or cloned DNA by PCR using labeled primers and are tethered between two optically trapped microspheres. We demonstrate that human, insect, plant, bacterial and viral sequences ranging from ~10 to 40 kilobasepairs can be manipulated. Force-extension measurements show that these constructs exhibit uniform elastic properties in accord with the expected contour lengths for the targeted sequences. Detailed protocols for preparing and manipulating these molecules are presented, and tethering efficiency is characterized as a function of DNA concentration, ionic strength and pH. Attachment strength is characterized by measuring the unbinding time as a function of applied force. An alternative stronger attachment method using an amino–carboxyl linkage, which allows for reliable DNA overstretching, is also described.


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