Nucleic Acids Research Advance Access originally published online on December 14, 2006
Nucleic Acids Research 2007 35(3):e16; doi:10.1093/nar/gkl1044
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Nucleic Acids Research, 2007, Vol. 35, No. 3 e16
© 2006 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 |
Rapid DNA mapping by fluorescent single molecule detection
1 Cardiovascular Research Institute and Center for Human Genetics, University of California San Francisco, CA 94115, USA 2 Department of Biochemistry and Biophysics, University of California San Francisco, CA 94115, USA 3 Department of Dermatology, University of California San Francisco, CA 94115, USA 4 Department of Physics and Center of Biophysics, University of Illinois at Urbana-Champaign Urbana, IL 61801, USA
*To whom correspondence should be addressed at: 513, Parnassus Avenue, HSW-901A, San Francisco, CA 94143, USA. Tel: +1 41 551 43876; Fax: +1 41 547 62956; Email: M.Xiao{at}ucsf.edu
Received October 13, 2006. Revised November 3, 2006. Accepted November 3, 2006.
DNA mapping is an important analytical tool in genomic sequencing, medical diagnostics and pathogen identification. Here we report an optical DNA mapping strategy based on direct imaging of individual DNA molecules and localization of multiple sequence motifs on the molecules. Individual genomic DNA molecules were labeled with fluorescent dyes at specific sequence motifs by the action of nicking endonuclease followed by the incorporation of dye terminators with DNA polymerase. The labeled DNA molecules were then stretched into linear form on a modified glass surface and imaged using total internal reflection fluorescence (TIRF) microscopy. By determining the positions of the fluorescent labels with respect to the DNA backbone, the distribution of the sequence motif recognized by the nicking endonuclease can be established with good accuracy, in a manner similar to reading a barcode. With this approach, we constructed a specific sequence motif map of lambda-DNA. We further demonstrated the capability of this approach to rapidly type a human adenovirus and several strains of human rhinovirus.
*Correspondence may also be addressed to Pui-Yan Kwok. Tel: +1 415-514-3802; Fax: +1 41 547 62956; Email: Pui.Kwok{at}ucsf.edu
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