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

  • Ravil R. GarafutdinovEmail author
  • Aidar R. Gilvanov
  • Assol R. Sakhabutdinova
Article
  • 42 Downloads

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.

Keywords

Nucleic acids Bst exo− DNA polymerase Isothermal amplification Nonspecific amplification Multimerization Rolling circle amplification 

Notes

Funding information

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

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of Biochemistry and Genetics, Ufa Federal Research CentreRussian Academy of SciencesUfaRussia

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