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Rapid DNA detection by beacon-assisted detection amplification

Nature Protocols volume 6, pages 772–778 (2011)Cite this article

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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.

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Figure 1: A schematic outline of the BAD AMP reaction described in the INTRODUCTION.
Figure 2: The DNA sequence of the molecular beacon containing a stem (blue), Nb.BbvCI endonuclease nicking site (orange), β-actin DNA target recognition sequence (black) along with a 6-FAM fluorophore (yellow star) and DABCYL quencher (black circle).
Figure 3: Replicate BAD AMP reactions were initiated with 1.7 pmol of the synthetic β-actin ssDNA target.
Figure 4: Replicate BAD AMP reactions were initiated with different amounts of the synthetic β-actin ssDNA target (A: 1.8 × 10−11 mol, B: 1.8 × 10−12 mol, C: 8.8 × 10−13 mol, D: 1.8 × 10−13 mol and E: 8.8 × 10−14 mol) or no DNA target (F).
Figure 5: Replicate BAD AMP reactions were initiated with different amounts of synthetic β-actin ssDNA target (1.8 × 10−12 mol, 8.8 × 10−13 mol, 1.8 × 10−13 mol and 8.8 × 10−14 mol) or no ssDNA target, as outlined in Table 1.
Figure 6: Replicate reactions containing 1.8 μg of heterogeneous RNA, the molecular beacon, Bst large fragment DNA polymerase and Nb.BvCI nicking endonuclease were initiated with different amounts of the synthetic β-actin ssDNA target (A: 1.8 × 10−11 mol, B: 1.8 × 10−12 mol, C: 1.8 × 10−13 mol and D: no synthetic DNA target).
Figure 7: The molecular beacon depicted in Figure 2 contained 16 bases complementary to β-actin DNA.

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Acknowledgements

We gratefully acknowledge funding and support from the NBCF through the National Collaborative Breast Cancer Research Grant.

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Authors and Affiliations

  1. Centre for Biomarker Research and Development, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St. Lucia, Queensland, Brisbane, Australia

    Ashley R Connolly & Matt Trau

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  1. Ashley R Connolly
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Contributions

A.R.C. conceived the idea. A.R.C. and M.T. designed and reviewed the entire experimental program. A.R.C. carried out the experiments and wrote the paper. All of the work was carried out within the laboratory of M.T.

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Correspondence to Ashley R Connolly.

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Connolly, A., Trau, M. Rapid DNA detection by beacon-assisted detection amplification. Nat Protoc 6, 772–778 (2011). https://doi.org/10.1038/nprot.2011.326

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