Rapid DNA detection by beacon-assisted detection amplification.

Ashley Connolly, Matt Trau

    Research output: Contribution to journalArticlepeer-review

    29 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)772-778
    Number of pages7
    JournalNature Protocols
    Volume6
    Issue number6
    DOIs
    Publication statusPublished - 2011

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