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Peptide nucleic acid molecular beacons for the detection of PCR amplicons in droplet-based microfluidic devices

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Abstract

The use of droplet-based microfluidics and peptide nucleic acid molecular beacons for the detection of polymerase chain reaction (PCR)-amplified DNA sequences within nanoliter-sized droplets is described in this work. The nanomolar–attomolar detection capabilities of the method were preliminarily tested by targeting two different single-stranded DNA sequences from the genetically modified Roundup Ready soybean and the Olea europaea genomes and detecting the fluorescence generated by peptide nucleic acid molecular beacons with fluorescence microscopy. Furthermore, the detection of 10 nM solutions of PCR amplicon of DNA extracted from leaves of O. europaea L. encapsulated in nanoliter-sized droplets was performed to demonstrate that peptide nucleic acid molecular beacons can discriminate O. europaea L. cultivar species carrying different single-nucleotide polymorphisms.

The fluorescence generated by peptide nucleic acid molecular beacons allows the detection of 100-200 attomoles of PCR-amplified DNA sequences from solutions encapsulated in nanoliter-sized droplets.

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Acknowledgments

We acknowledge support from MIUR (PRIN 20093N774P, FIRB RBRN07BMCT, and RBPR05JH2P ITALNANONET).

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Author notes

  1. Marco Licciardello

    Present address: Center for Molecular Medicine of the Austrian Academy of Sciences, Lazarettgasse 14, 1090, Vienna, Austria

Authors and Affiliations

  1. Scuola Superiore di Catania, c/o Dipartimento di Scienze Chimiche, Università di Catania, Viale Andrea Doria 6, 95125, Catania, Italy

    Laura Maria Zanoli

  2. Dipartimento di Scienze Chimiche, Università di Catania, Viale Andrea Doria 6, 95125, Catania, Italy

    Marco Licciardello, Roberta D’Agata & Giuseppe Spoto

  3. Dipartimento di Chimica Organica e Industriale, Università di Parma, V.le G. P. Usberti 17/a, 43100, Parma, Italy

    Claudia Lantano, Alessandro Calabretta, Roberto Corradini & Rosangela Marchelli

  4. Istituto Biostrutture e Bioimmagini, CNR, Viale A. Doria 6, 95125, Catania, Italy

    Giuseppe Spoto

Authors
  1. Laura Maria Zanoli
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  2. Marco Licciardello
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  7. Rosangela Marchelli
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Corresponding author

Correspondence to Giuseppe Spoto.

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Published in the special issue Analytical Science in Italy with guest editor Aldo Roda.

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Zanoli, L.M., Licciardello, M., D’Agata, R. et al. Peptide nucleic acid molecular beacons for the detection of PCR amplicons in droplet-based microfluidic devices. Anal Bioanal Chem 405, 615–624 (2013). https://doi.org/10.1007/s00216-011-5638-3

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  • DOI: https://doi.org/10.1007/s00216-011-5638-3

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