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Synthesis of circular DNA templates with T4 RNA ligase for rolling circle amplification

  • Structural and Functional Analysis of Biopolymers and Their Complexes
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Abstract

Currently, isothermal methods of nucleic acid amplification have been well established; in particular, rolling circle amplification is of great interest. In this approach, circular ssDNA molecules have been used as a target that can be obtained by the intramolecular template-dependent ligation of an oligonucleotide C-probe. Here, a new method of synthesizing small circular DNA molecules via the cyclization of ssDNA based on T4 RNA ligase has been proposed. Circular ssDNA is further used as the template for the rolling circle amplification. The maximum yield of the cyclization products was observed in the presence of 5−10% polyethylene glycol 4000, and the optimum DNA length for the cyclization constituted 50 nucleotides. This highly sensitive method was shown to detect less than 102 circular DNA molecules. The method reliability was proved based on artificially destroyed dsDNA, which suggests its implementation for analyzing any significantly fragmented dsDNA.

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Abbreviations

PCR:

polymerase chain reaction

RCA:

rolling circle amplification

NA:

nucleic acid

PEG:

polyethylene glycol

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Authors and Affiliations

  1. Institute of Biochemistry and Genetics, Ufa Science Centre, Russian Academy of Sciences, Ufa, 450054, Russia

    A. R. Sakhabutdinova, M. A. Maksimova & R. R. Garafutdinov

Authors
  1. A. R. Sakhabutdinova
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  2. M. A. Maksimova
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  3. R. R. Garafutdinov
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Correspondence to A. R. Sakhabutdinova.

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Original Russian Text © A.R. Sakhabutdinova, M.A. Maksimova, R.R. Garafutdinov, 2017, published in Molekulyarnaya Biologiya, 2017, Vol. 51, No. 4, pp. 724–733.

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Sakhabutdinova, A.R., Maksimova, M.A. & Garafutdinov, R.R. Synthesis of circular DNA templates with T4 RNA ligase for rolling circle amplification. Mol Biol 51, 639–646 (2017). https://doi.org/10.1134/S0026893317040161

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  • DOI: https://doi.org/10.1134/S0026893317040161

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