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Intramolecular Interactions in the Fluorophore–Quencher System in Linear and Hairpin Probes for Real-Time PCR

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Abstract

The hairpin probe is characterized by a higher fluorescence quenching efficiency as compared with the linear one under the conditions of real-time PCR, which leads to a lower background level of fluorescence and, consequently, a higher signal to noise ratio during real-time PCR. An experimental comparison of the fluorescence-quenching efficiency of two oligonucleotide probes in different conformations (hairpin in a molecular beacon format and linear in a TaqMan format) was made. There is a difference in the interaction between the quencher and fluorophore for the probes of different conformations. For a linear probe, quenching occurs through the mechanism of inductive-resonance energy transfer (Förster resonance energy transfer, FRET), while that for a hairpin probe quenching occurs through contact quenching through a closer arrangement of the fluorophore and quencher, but a resonant energy transfer according to the Förster mechanism is also possible. It was demonstrated that the absorption spectrum for the linear probe almost coincides with the absorption spectrum of an oligonucleotide representing a probe without a quencher, which indicates a dynamic (Förster) mechanism of energy transfer. On the contrary, the absorption spectra for the hairpin probe and oligonucleotide representing the probe without a quencher differ significantly, which indicates a contact mechanism of energy transfer between the fluorophore and fluorescence quencher. The fluorescence spectra of the probes and their complexes with the oligonucleotide complementary to the linear probe (and the loop of the hairpin probe) and the amplicon (200 bp in length containing a DNA target for the probes) allowed for comparison of these two probes by comparing the energy migration radii, the efficiency of donor fluorescence quenching. The energy migration radius R calculated by the experimental data was 32.4 Å for the hairpin probe and 47.3 Å for the linear probe.

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ACKNOWLEDGMENTS

We are grateful to L.A. Murtazaeva for the assistance in performing spectral studies and to Doctor in Physics and Mathematics, Professor of Karazin Kharkiv National University G.P. Gorbenko.

Funding

This study was supported by Doctor in Biology O.Yu. Limanskaya and Doctor in Biology O.P. Limanskii without the external funding.

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Authors and Affiliations

  1. National Scientific Center Institute of Experimental and Clinical Veterinary Medicine, National Academy of Agrarian Sciences of Ukraine, 61023, Kharkiv, Ukraine

    O. Yu. Limanskaya

  2. Institute of Physiologically Active Compounds, 61072, Kharkiv, Ukraine

    O. P. Limanskii

Authors
  1. O. Yu. Limanskaya
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  2. O. P. Limanskii
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Corresponding authors

Correspondence to O. Yu. Limanskaya or O. P. Limanskii.

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The authors declare that they have no conflicts of interest. This article does not contain any studies involving human participants and animals as objects.

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Limanskaya, O.Y., Limanskii, O.P. Intramolecular Interactions in the Fluorophore–Quencher System in Linear and Hairpin Probes for Real-Time PCR. Cytol. Genet. 57, 134–141 (2023). https://doi.org/10.3103/S009545272302007X

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  • DOI: https://doi.org/10.3103/S009545272302007X

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