title: Sequence specific detection of DNA using nicking endonuclease signal amplification (NESA).
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology & Cellular Biology, Georgetown University School of Medicine, Washington, DC 20057, USA.
authors.forename: Traci
authors.initials: T
authors.lastname: Kiesling
authors.forename: Kendra
authors.initials: K
authors.lastname: Cox
authors.forename: Eugene A
authors.initials: EA
authors.lastname: Davidson
authors.forename: Kenneth
authors.initials: K
authors.lastname: Dretchen
authors.forename: Guy
authors.initials: G
authors.lastname: Grater
authors.forename: Shannon
authors.initials: S
authors.lastname: Hibbard
authors.forename: Roger S
authors.initials: RS
authors.lastname: Lasken
authors.forename: Jonathan
authors.initials: J
authors.lastname: Leshin
authors.forename: Evan
authors.initials: E
authors.lastname: Skowronski
authors.forename: Mark
authors.initials: M
authors.lastname: Danielsen
mesh_terms: D001408:Bacillus anthracis / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis; D015252:Deoxyribonucleases, Type II Site-Specific; D019075:Electrophoresis, Capillary; D007700:Kinetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature