title: Rapid DNA detection by beacon-assisted detection amplification.
abstract: This protocol describes a new and rapid isothermal reaction process designed to amplify and detect a specific DNA sequence in purified DNA extracted from cultured cells. The protocol uses a DNA nanomachine that comprises two molecular switches that function in concert to isothermally amplify and detect a DNA target. First, a molecular beacon detection switch is 'activated' only if a DNA target sequence is present. A DNA primer and DNA polymerase are used to lock the beacon in an activated conformation. Second, an amplification and signal-transduction switch is initiated following successful activation. A nicking endonuclease and the DNA polymerase are used to replicate the DNA target. Both switches operate simultaneously at 40 °C in a single reaction to rapidly generate multiple copies of the DNA target in a cyclic polymerization reaction. This protocol enables femtomole amounts of a DNA target to be reproducibly amplified and detected in <40 min. We demonstrate the successful use of this protocol in assays containing synthetic DNA components and purified DNA extracted from biological samples.
authors.affiliation: Centre for Biomarker Research and Development, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St. Lucia, Brisbane, Queensland, Australia. a.connolly@uq.edu.au
authors.forename: Ashley R
authors.initials: AR
authors.lastname: Connolly
authors.forename: Matt
authors.initials: M
authors.lastname: Trau
mesh_terms: D000199:Actins / Q000737:chemistry / Q000235:genetics; D004247:DNA / Q000737:chemistry*; D005453:Fluorescence; D021141:Nucleic Acid Amplification Techniques; D017422:Sequence Analysis, DNA
chemical_list: D000199:Actins; D004247:DNA