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


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.


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


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.


  1. 1.
    Mullis, K. B., & Faloona, F. A. (1987). Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology, 155, 335–350.CrossRefGoogle Scholar
  2. 2.
    Bartlett, J. M. S., & Stirling, D. (2003). Methods in molecular biology, vol. 226: PCR protocols (2nd ed.). Totowa: Humana.Google Scholar
  3. 3.
    Demidov, V. V., & Broude, N. E. (2004). DNA amplification: current technologies and applications (1st ed.). Wymondham: Horizon bioscience.Google Scholar
  4. 4.
    Fakruddin, M., Mannan, K. S., Chowdhury, A., Mazumdar, R. M., Hossain, M. N., Islam, S., & Chowdhury, M. A. (2013). Nucleic acid amplification: alternative methods of polymerase chain reaction. Journal of Pharmacy and Bioallied Sciences, 5(4), 245–252.CrossRefGoogle Scholar
  5. 5.
    Zhao, Y., Chen, F., Li, Q., Wang, L., & Fan, C. (2015). Isothermal amplification of nucleic acids. Chemical reviews, 115(22), 12491–12545.CrossRefGoogle Scholar
  6. 6.
    Fozooni, T., Ravan, H., & Sasan, H. (2017). Signal amplification technologies for the detection of nucleic acids: from cell-free analysis to live-cell imaging. Applied Biochemistry and Biotechnology, 183(4), 1224–1253.CrossRefGoogle Scholar
  7. 7.
    Ko, J., & Yoo, J. C. (2018). Loop-mediated isothermal amplification using a lab-on-a-disc device with thin-film phase change material. Applied Biochemistry and Biotechnology, 186(1), 54–65.CrossRefGoogle Scholar
  8. 8.
    Chen, Y., Cheng, N., Xu, Y., Huang, K., Luo, Y., & Xu, W. (2016). Point-of-care and visual detection of P. aeruginosa and its toxin genes by multiple LAMP and lateral flow nucleic acid biosensor. Biosensors and Bioelectronics, 81, 317–323.CrossRefGoogle Scholar
  9. 9.
    Moghimi, H., Moradi, A., Hamedi, J., & Basiri, M. (2015). Development of a loop-mediated isothermal amplification assay for rapid and specific identification of ACT producing Alternaria alternata, the agent of brown spot disease in tangerine. Applied Biochemistry and Biotechnology, 178(6), 1207–1219.CrossRefGoogle Scholar
  10. 10.
    Kaocharoen, S., Wang, W., Tsui, K. M., Trilles, L., Kong, F., & Meyer, W. (2008). Hyperbranched rolling circle amplification as a rapid and sensitive method for species identification within the Cryptococcus species complex. Electrophoresis, 29(15), 3183–3191.Google Scholar
  11. 11.
    Wang, X. R., Wu, L. F., Wang, Y., Ma, Y. Y., Chen, F. H., & Ou, H. L. (2015). Rapid detection of Staphylococcus aureus by loop-mediated isothermal amplification. Applied Biochemistry and Biotechnology, 175(2), 882–891.CrossRefGoogle Scholar
  12. 12.
    Liu, W., Zhang, H., Hu, D., Lu, S., & Sun, X. (2018). The performance of MALBAC and MDA methods in the identification of concurrent mutations and aneuploidy screening to diagnose beta-thalassaemia disorders at the single- and multiple-cell levels. Journal of Clinical Laboratory Analysis, 32(2), e22267.CrossRefGoogle Scholar
  13. 13.
    Tao, C., Yang, Y., Li, X., Zheng, X., Ren, H., Li, K., & Zhou, R. (2016). Rapid and sensitive detection of sFAT-1 transgenic pigs by visual loop-mediated isothermal amplification. Applied Biochemistry and Biotechnology, 179(6), 938–946.CrossRefGoogle Scholar
  14. 14.
    Lv, J., Xie, S., Cai, W., Zhang, J., Tang, D., & Tang, Y. (2017). Highly effective target converting strategy for ultrasensitive electrochemical assay of Hg2+. The Analyst, 142(24), 4708–4714.CrossRefGoogle Scholar
  15. 15.
    Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., & Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28(12), E63.CrossRefGoogle Scholar
  16. 16.
    Compton, J. (1991). Nucleic acid sequence-based amplification. Nature, 350(6313), 91–92.CrossRefGoogle Scholar
  17. 17.
    Walter, N. G., & Strunk, G. (1994). Strand displacement amplification as an in vitro model for rolling-circle replication: deletion formation and evolution during serial transfer. Proceedings of the National Academy of Sciences of the United States of America, 91(17), 7937–7941.CrossRefGoogle Scholar
  18. 18.
    Fire, A., & Xu, S. Q. (1995). Rolling replication of short DNA circles. Proceedings of the National Academy of Sciences of the United States of America, 92(10), 4641–4645.CrossRefGoogle Scholar
  19. 19.
    Mohsen, M. G., & Kool, E. T. (2016). The discovery of rolling circle amplification and rolling circle transcription. Accounts of Chemical Research, 49(11), 2540–2550.CrossRefGoogle Scholar
  20. 20.
    Ali, M. M., Li, F., Zhang, Z., Zhang, K., Kang, D. K., Ankrum, J. A., Le, X. C., & Zhao, W. (2014). Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine. Chemical Society Reviews, 43(10), 3324–3341.CrossRefGoogle Scholar
  21. 21.
    Murakami, T., Sumaoka, J., & Komiyama, M. (2009). Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification. Nucleic Acids Research, 37(3), e19.CrossRefGoogle Scholar
  22. 22.
    Gu, L., Yan, W., Liu, L., Wang, S., Zhang, X., & Lyu, M. (2018). Research progress on rolling circle amplification (RCA)-based biomedical sensing. Pharmaceuticals, 11(2), 35.CrossRefGoogle Scholar
  23. 23.
    Oscorbin, I. P., Boyarskikh, U. A., & Filipenko, M. L. (2015). Large fragment of DNA polymerase I from Geobacillus sp. 777: cloning and comparison with DNA polymerases I in practical applications. Molecular Biotechnology, 57(10), 947–959.CrossRefGoogle Scholar
  24. 24.
    Oscorbin, I. P., Belousova, E. A., Boyarskikh, U. A., Zakabunin, A. I., Khrapov, E. A., & Filipenko, M. L. (2017). Derivatives of Bst-like Gss-polymerase with improved processivity and inhibitor tolerance. Nucleic Acids Research, 45(16), 9595–9610.CrossRefGoogle Scholar
  25. 25.
    Zyrina, N. V., Antipova, V. N., & Zheleznaya, L. A. (2014). Ab initio synthesis by DNA polymerases. FEMS Microbiology Letters, 351(1), 1–6.CrossRefGoogle Scholar
  26. 26.
    Hafner, G. J., Yang, I. C., Wolter, L. C., Stafford, M. R., & Giffard, P. M. (2001). Isothermal amplification and multimerization of DNA by Bst DNA polymerase. BioTechniques, 30(4), 852–867.CrossRefGoogle Scholar
  27. 27.
    Wang, G., Ding, X., Hu, J., Wu, W., Sun, J., & Mu, Y. (2017). Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities. Scientific Reports, 7(1), 13928.CrossRefGoogle Scholar
  28. 28.
    Viguera, E., Canceill, D., & Ehrlich, S. D. (2001). In vitro replication slippage by DNA polymerases from thermophilic organisms. Journal of Molecular Biology, 312(2), 323–333.CrossRefGoogle Scholar
  29. 29.
    Qian, J., Ferguson, T. M., Shinde, D. N., Ramírez-Borrero, A. J., Hintze, A., Adami, C., & Niemz, A. (2012). Sequence dependence of isothermal DNA amplification via EXPAR. Nucleic Acids Research, 40(11), e87.CrossRefGoogle Scholar
  30. 30.
    Sambrook, J., & Russell, D. W. (2006). Isolation of DNA fragments from polyacrylamide gels by the crush and soak method. CSH Protocols., 2006(1), pdb.prot2936. Scholar
  31. 31.
    Güixens-Gallardo, P., Hocek, M., & Perlíková, P. (2016). Inhibition of non-templated nucleotide addition by DNA polymerases in primer extension using twisted intercalating nucleic acid modified templates. Bioorganic & Medicinal Chemistry Letters, 26(2), 288–291.CrossRefGoogle Scholar
  32. 32.
    Lee, D., Shin, Y., Seok, C., Hwang, K. S., Yoon, D. S., & Lee, J. H. (2016). Simple and Highly sensitive molecular diagnosis of Zika Virus by lateral flow analysis. Analytical Chemistry, 88(24), 12272–12278.CrossRefGoogle Scholar
  33. 33.
    Oscorbin, I. P., Belousova, E. A., Zakabunin, A. I., Boyarskikh, U. A., & Filipenko, M. L. (2016). Comparison of fluorescent intercalating dyes for quantitative loop-mediated isothermal amplification (qLAMP). Biotechniques., 61, 20–25.CrossRefGoogle Scholar

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Authors and Affiliations

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

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