The Influence of Reaction Conditions on DNA Multimerization During Isothermal Amplification with Bst exo- DNA...

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.