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Structural Destabilization of Intramolecular Duplexes Improves the Results of DNA Hybridization Analysis

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Abstract

This study tested a method designed to correctly identify single nucleotide polymorphisms in DNA sequences that are capable of forming a hairpin. Fragments of the angiotensin (AGT) and cytochrome (CYP2C19) genes with rs699 (T>C) and rs4986893 (G>A), respectively, were chosen as examples. DNA probes complementary to the polymorphic sites formed hairpin structures with a loop of 6 nt in the case of rs699 (AGT) or 4 nt in the case of rs4986893 (CYP2C19). Fluorophore-labeled target DNA was obtained via two-round multiplex PCR with simultaneous incorporation of a fluorescent label in the second round. When target DNA was hybridized to a corresponding pair of probes immobilized in gel pads of a biochip, dramatically low, if any, fluorescent signals were detected from the pads. A replacement of one nucleotide in the DNA probes prevented the formation of intramolecular structures, as was confirmed by melting curves. However, the DNA probes completely lost their complementarity to target DNA as a result of the replacement. To restore the complementary interaction with the DNA probe, corresponding nucleotide replacements were introduced in target DNA via site-directed mutagenesis. The approach significantly increased the specific fluorescent signals from biochip pads, thus allowing correct genotyping of rs699 and rs4986893.

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ACKNOWLEDGMENTS

This work was supported by the Studies and Developments in Priority Fields of the Research and Technology Complex of Russia from 2014 to 2020 federal program (agreement no. 14.604.21.0166, unique project identifier RFMEFI60417X0166).

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Authors and Affiliations

  1. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991, Moscow, Russia

    A. Yu. Ikonnikova, O. A. Zasedateleva, S. A. Surzhikov, V. O. Pozhitnova, D. O. Fesenko, A. A. Stomakhin, A. S. Zasedatelev & T. V. Nasedkina

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  1. A. Yu. Ikonnikova
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  2. O. A. Zasedateleva
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  3. S. A. Surzhikov
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  4. V. O. Pozhitnova
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  5. D. O. Fesenko
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  6. A. A. Stomakhin
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  7. A. S. Zasedatelev
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  8. T. V. Nasedkina
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Corresponding author

Correspondence to T. V. Nasedkina.

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Conflict of interests. The authors declare that they have no conflict of interest.

Statement of compliance with standards of research involving humans as subjects. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants involved in the study.

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Translated by T. Tkacheva

Abbreviations: PCR, polymerase chain reaction; bp, base pair.

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Ikonnikova, A.Y., Zasedateleva, O.A., Surzhikov, S.A. et al. Structural Destabilization of Intramolecular Duplexes Improves the Results of DNA Hybridization Analysis. BIOPHYSICS 63, 880–887 (2018). https://doi.org/10.1134/S000635091806012X

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  • DOI: https://doi.org/10.1134/S000635091806012X

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