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Nucleic Acids Research 2005 33(18):5829-5837; doi:10.1093/nar/gki895
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Published online 20 October 2005

© The Author 2005. 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

Article

Application of single molecule technology to rapidly map long DNA and study the conformation of stretched DNA

Kevin M. Phillips, Jonathan W. Larson, Gregory R. Yantz, Christina M. D'Antoni, Michael V. Gallo, Kimberly A. Gillis, Nuno M. Goncalves, Lori A. Neely, Steven R. Gullans and Rudolf Gilmanshin*

U. S. Genomics, Inc. 12 Gill Street, Suite 4700, Woburn, MA 01801, USA

*To whom correspondence should be addressed. Tel: +781 939 6404; Fax: +781 938 0060; Email: rgilmanshin{at}usgenomics.com

Received March 29, 2005. Revised June 10, 2005. Accepted September 27, 2005.

Herein we describe the first application of direct linear analysis (DLA) to the mapping of a bacterial artificial chromosome (BAC), specifically the 185.1 kb-long BAC 12M9. DLA is a single molecule mapping technology, based on microfluidic elongation and interrogation of individual DNA molecules, sequence-specifically tagged with bisPNAs. A DNA map with S/N ratio sufficiently high to detect all major binding sites was obtained using only 200 molecule traces. A new method was developed to extract an oriented map from an averaged map that included a mixture of head-first and tail-first DNA traces. In addition, we applied DLA to study the conformation and tagging of highly stretched DNA. Optimal conditions for promoting sequence-specific binding of bisPNA to an 8 bp target site were elucidated using DLA, which proved superior to electromobility shift assays. DLA was highly reproducible with a hybridized tag position localized with an accuracy of ±0.7 µm or ±2.1 kb demonstrating its utility for rapid mapping of large DNA at the single molecule level. Within this accuracy, DNA molecules, stretched to at least 85% of their contour length, were stretched uniformly, so that the map expressed in relative coordinates, was the same regardless of the molecule extension.

Present address: Kimberly A. Gillis, Baxter Healthcare Corporation, 220 Norwood Park South, Norwood, MA 02062, USA


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