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Two-dye and one- or two-quencher DNA probes for real-time PCR assay: synthesis and comparison with a TaqMan™ probe

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Abstract

A typical TaqMan™ real-time PCR probe contains a 5′-fluorescent dye and a 3′-quencher. In the course of the amplification, the probe is degraded starting from the 5′-end, thus releasing fluorescent dye. Some fluorophores (including fluorescein) are known to be prone to self-quenching when located near each other. This work is aimed at studying dye–dye and dye–quencher interactions in multiply modified DNA probes. Twenty-one fluorogenic probes containing one and two fluoresceins (FAM), or a FAM–JOE pair, and one or two BHQ1 quenchers were synthesized using non-nucleoside reagents and “click chemistry” post-modification on solid phase and in solution. The probes were tested in real-time PCR using an ~300-bp-long natural DNA fragment as a template. The structural prerequisites for lowering the probe background fluorescence and increasing the end-plateau fluorescence intensity were evaluated and discussed.

Fluorogenic TaqMan probes with various modifications for real-time PCR

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Acknowledgments

The research was supported by the Molecular and Cellular Biology Program of the Russian Academy of Sciences, Russian Foundation for Basic Research (project no. 10-04-00998), and the Ministry of Industry and Trade of the Russian Federation (contract no. 11411.0810200.13.B24). The authors are grateful to Irina A. Stepanova and Elena V. Nozhevnikova for their help in data analysis and Tatyana E. Chernichko for the reading of the manuscript.

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Authors and Affiliations

  1. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997, Moscow, Russia

    Dmitry Y. Ryazantsev, Igor A. Prokhorenko, Vladimir A. Korshun & Sergey K. Zavriev

  2. Institute of Physical Organic Chemistry, Surganova 13, 220072, Minsk, Belarus

    Dmitry A. Tsybulsky, Maksim V. Kvach, Yury V. Martynenko & Vadim V. Shmanai

  3. Primetech LLC, Surganova 13, 220072, Minsk, Belarus

    Pavel M. Philipchenko

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  1. Dmitry Y. Ryazantsev
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  2. Dmitry A. Tsybulsky
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  3. Igor A. Prokhorenko
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  4. Maksim V. Kvach
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  5. Yury V. Martynenko
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  6. Pavel M. Philipchenko
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  7. Vadim V. Shmanai
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  8. Vladimir A. Korshun
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  9. Sergey K. Zavriev
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Correspondence to Vladimir A. Korshun or Sergey K. Zavriev.

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Ryazantsev, D.Y., Tsybulsky, D.A., Prokhorenko, I.A. et al. Two-dye and one- or two-quencher DNA probes for real-time PCR assay: synthesis and comparison with a TaqMan™ probe. Anal Bioanal Chem 404, 59–68 (2012). https://doi.org/10.1007/s00216-012-6114-4

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  • DOI: https://doi.org/10.1007/s00216-012-6114-4

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