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

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 pi...

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Bibliographic Details
Published in:Nucleic acids research 2007-09, Vol.35 (18), p.e117-e117
Main Authors: Kiesling, Traci, Cox, Kendra, Davidson, Eugene A, Dretchen, Kenneth, Grater, Guy, Hibbard, Shannon, Lasken, Roger S, Leshin, Jonathan, Skowronski, Evan, Danielsen, Mark
Format: Article
Language:English
Subjects:
Bacillus
Bacillus anthracis - genetics
Bacillus subtilis
Bacillus subtilis - genetics
Deoxyribonucleases, Type II Site-Specific
DNA, Bacterial - analysis
Electrophoresis, Capillary
Kinetics
Methods Online
Nucleic Acid Hybridization - methods
Oligonucleotide Probes
Polymerase Chain Reaction
Sequence Analysis, DNA - methods
Temperature
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Summary: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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkm654