Nucleic Acids Research Advance Access originally published online on September 7, 2007
Nucleic Acids Research 2007 35(18):e117; doi:10.1093/nar/gkm654
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Nucleic Acids Research, 2007, Vol. 35, No. 18 e117
© 2007 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.
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Sequence specific detection of DNA using nicking endonuclease signal amplification (NESA)
1Department of Biochemistry and Molecular Biology & Cellular Biology, 2Department of Pharmacology, Georgetown University School of Medicine, Washington DC 20057, 3Anteon Corporation, Fairfax, VA 22030, 4J. Craig Venter Institute, La Jolla, CA 92037 and 5General Dynamics, Fairfax, VA 22030, USA
*To whom correspondence should be addressed. Tel: +1 202 687 4169; Fax: +1 202 687 7186; Email: dan{at}bc.georgetown.edu
Received May 8, 2007. Revised August 5, 2007. Accepted August 8, 2007.
We have developed a new method for identifying specific single- or double-stranded DNA sequences called nicking endonuclease signal amplification (NESA). A probe and target DNA anneal to create a restriction site that is recognized by a strand-specific endonuclease that cleaves the probe into two pieces leaving the target DNA intact. The target DNA can then act as a template for fresh probe and the process of hybridization, cleavage and dissociation repeats. Laser-induced fluorescence coupled with capillary electrophoresis was used to measure the probe cleavage products. The reaction is rapid; full cleavage of probe occurs within one minute under ideal conditions. The reaction is specific since it requires complete complementarity between the oligonucleotide and the template at the restriction site and sufficient complementarity overall to allow hybridization. We show that both Bacillus subtilis and B. anthracis genomic DNA can be detected and specifically differentiated from DNA of other Bacillus species. When combined with multiple displacement amplification, detection of a single copy target from less than 30 cfu is possible. This method should be applicable whenever there is a requirement to detect a specific DNA sequence. Other applications include SNP analysis and genotyping. The reaction is inherently simple to multiplex and is amenable to automation.
Present addresses: Evan Skowronski, US Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD 21010 Kendra Cox, Cepheid CA 94089 Guy Grater, Alion Science and Technology Corporation, McLean, VA 22102 Shannon Hibbard, Analytic Services Inc., Arlington, VA 22206 Traci Kiesling, Booz Allen Hamilton Inc, McLean, VA 22102.
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