Abstract
In recent years, methods of isothermal amplification of nucleic acids using polymerases with a strand-displacement activity have become widespread for identification of specific nucleotide sequences. Bst exo– polymerase is the most popular of these polymerases, although it is inclined to nonspecific amplification (the so-called multimerization), which leads to the accumulation of by-products constructed of tandem nucleotide repeats. In this study, we evaluated the efficiency of multimerization depending on the reaction conditions and proposed some methods for its elimination. The highest efficiency of multimerization was found in the case of Bst 2.0 polymerase in Isothermal buffer, whereas the Bst-like Gss polymerase provided the formation of multimerization products only in Isothermal buffer and at the latest stages of the reaction. The optimal method for elimination of multimerization was the use of Gss polymerase and Thermopol buffer, or Bst LF polymerase and Isothermal II buffer, or Bst 3.0 polymerase and Thermopol buffer, or Bst 3.0 polymerase in Isothermal buffer and Mn2+ ions as a cofactor. In these cases specific isothermal amplification of the target DNA may take place and provide accurate and reliable results.
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ACKNOWLEDGMENTS
The studies were conducted using the equipment of the Center of Collective Use Biomika and a unique scientific system KODINK.
Funding
The work was supported by Russian State Federal budget (project no. АААА-А16-116020350032-1) and partially supported by Russian State-funded Budget project no. АААА-А17-117020210023-1 to ICBFM SB RAS.
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In this work, humans or animals were not involved as subjects of studies.
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Translated by E. Shirokova
Abbreviations: CT, circular DNA templates; DMTr, dimethoxytrityl; DTT, dithiothreitol; FAM, 5'-carboxyfluoresceine; LT, linear single-stranded DNA templates; RCA, rolling circle amplification.
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Sakhabutdinova, A.R., Mirsaeva, L.R., Oscorbin, I.P. et al. Elimination of DNA Multimerization Arising from Isothermal Amplification in the Presence of Bst Exo– DNA Polymerase. Russ J Bioorg Chem 46, 52–59 (2020). https://doi.org/10.1134/S1068162020010082
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DOI: https://doi.org/10.1134/S1068162020010082