Biomics

The nucleic acids amplification is of great significance in the genomic research and DNA diagnostics. It is carried out by different techniques, in the first place by polymerase chain reaction. Recently, isothermal methods of amplification, e.g. rolling circle amplification, are becoming more common. For these techniques, DNA polymerases with strand-displacing activity are used. Among these enzymes Bst DNA polymerase is the most popular, but it is characterized by the formation of nonspecific products. In this work the influence of SYBR Green I intercalating dye concentration on the formation of specific and nonspecific products during rolling circle amplification by Bst polymerase large fragment (Bst exo^-) has been studied. The optimal SYBR Green I concentrations which completely inhibit the formation of multimeric products on the linear templates and almost have no affect on the amplification of the circular templates have been determined.

isothermal amplification; multimerization; Bst exo DNA polymerase; SYBR Green I; inhibition

1. Ali M.M., Li F., Zhang Zh., 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. Soc. Rev. V. 43. P. 3324-3341. doi: 10.1039/c3cs60439j
2. Deng H., Gao Z. Bioanalytical applications of isothermal nucleic acid amplification techniques. Chim. Acta. 2015. V. 853. P. 30-45. doi: 10.1016/j.aca.2014.09.037
3. Fakruddin M., Mannan K.S., Chowdhury A., Mazumdar R.M., Hossain M. N., Islam S., Chowdhury M.A. Nucleic acid amplification: Alternative methods of polymerase chain reaction. Pharm. Bioall. Sci. 2013. V. 5(4). P. 245-252. doi: 10.4103/0975-7406.120066
4. Gudnason H., Dufva M., Bang D.D., Wolff A. Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Res. 2007. V. 35(19). e127. doi: 10.1093/nar/gkm671
5. Hafner G.J, Yang I.C., Wolter L.C., Stafford M.R., Giffard P.M. Isothermal amplification and multimerization of DNA by Bst DNA polymerase. BioTechniques. 2001. V. 30(4), P. 852-86.
6. Monis P.T., Giglio S., Saint P. C. Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis. Biochem. 2005. V. 340(1). P. 24-34. doi: 10.1016/j.ab.2005.01.046
7. Sakhabutdinova A.R., Maksimova, M.A., Garafutdinov R.R. Synthesis of Circular DNA Templates with T4 RNA Ligase for Rolling Circle Amplification. Molecular biology. 2017. V. 51(4). P. 639-646. doi: 10.7868/S0026898417040164
8. van Oorschot R.A.H., Ballantyne K.N., Mitchell R.J. Forensic trace DNA: a review. Investigative Genetics. 2010. V. 1 (14). doi: 10.1186/2041-2223-1-14
9. Wang, Ding X., Hu J., Wu W., Sun J., Mu Y. Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities. Sci. Rep. 2017. V. 7. article number:13928. doi: 10.1038/s41598-017-13324-0
10. Zhao Y., Chen F., Li Q., Wang L., Fan C. Isothermal amplification of nucleic acids. Rev. 2015. V. 115. P.12491-12545. doi: 10.1021/acs.chemrev.5b00428