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The Influence of Reaction Conditions on DNA Multimerization During Isothermal Amplification with Bst exo− DNA Polymerase

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Abstract

Methods for isothermal amplification of nucleic acids are gained more attention in the last two decades. For isothermal amplification, DNA polymerases with strand displacement activity are required, and Bst exo− is one of the most commonly used polymerases. However, Bst exo− is able to cause nonspecific DNA amplification through multimerization, which leads to a set of undesirable by-products. In this study, circumstances that facilitate DNA multimerization by Bst exo− polymerase have been determined. We found that an essential requirement for multimerization is the presence of short (50–60 bp) DNA duplexes formed through primer extension after annealing on the template or in homo- and heterodimers. The highest multimerization efficiency is observed for Bst 2.0 polymerase in buffers with a high salt concentration and/or in the presence of reducing agents (for example, β-mercaptoethanol). Multimerization occurs mainly at 55–60 °С, while specific isothermal amplification is more efficient at 60–65 °С. The SYBR Green I intercalating dye inhibits multimerization with Bst LF and Bst 2.0 polymerases in concentrations above 0.25×, whereas inhibition with Bst 3.0 polymerase occurs only above 1.25×. The obtained results allow to elaborate accurate and reliable methods for isothermal amplification of nucleic acids.

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Funding

This work was supported by the Russian State Federal budget (No. АААА-А16-116020350032-1).

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  1. Institute of Biochemistry and Genetics, Ufa Federal Research Centre, Russian Academy of Sciences, prosp. Oktyabrya, 71, Ufa, Bashkortostan, Russia, 450054

    Ravil R. Garafutdinov, Aidar R. Gilvanov & Assol R. Sakhabutdinova

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  1. Ravil R. Garafutdinov
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  2. Aidar R. Gilvanov
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Correspondence to Ravil R. Garafutdinov.

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Garafutdinov, R.R., Gilvanov, A.R. & Sakhabutdinova, A.R. The Influence of Reaction Conditions on DNA Multimerization During Isothermal Amplification with Bst exo− DNA Polymerase. Appl Biochem Biotechnol 190, 758–771 (2020). https://doi.org/10.1007/s12010-019-03127-6

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