Skip Navigation



Nucleic Acids Research Advance Access published online on September 7, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm654
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (192K) Freely available
Right arrow Screen PDF (202K) Freely available
Right arrowOA All Versions of this Article:
35/18/e117    most recent
gkm654v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Kiesling, T.
Right arrow Articles by Danielsen, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kiesling, T.
Right arrow Articles by Danielsen, M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 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.

Methods Online

Sequence specific detection of DNA using nicking endonuclease signal amplification (NESA)

Traci Kiesling1, Kendra Cox2, Eugene A. Davidson1, Kenneth Dretchen2, Guy Grater3, Shannon Hibbard2, Roger S. Lasken4, Jonathan Leshin2, Evan Skowronski5 and Mark Danielsen1,*

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.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
J. J. Li, Y. Chu, B. Y.-H. Lee, and X. S. Xie
Enzymatic signal amplification of molecular beacons for sensitive DNA detection
Nucleic Acids Res., April 1, 2008; 36(6): e36 - e36.
[Abstract] [Full Text] [PDF]


Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.