Molecular Biology

, Volume 51, Issue 4, pp 639–646 | Cite as

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

  • A. R. Sakhabutdinova
  • M. A. Maksimova
  • R. R. Garafutdinov
Structural and Functional Analysis of Biopolymers and Their Complexes
  • 60 Downloads

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.

Keywords

rolling circle amplification ramification circular DNA T4 RNA ligase intramolecular template-independent ligation 

Abbreviations

PCR

polymerase chain reaction

RCA

rolling circle amplification

NA

nucleic acid

PEG

polyethylene glycol

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Copyright information

© Pleiades Publishing, Inc. 2017

Authors and Affiliations

  • A. R. Sakhabutdinova
    • 1
  • M. A. Maksimova
    • 1
  • R. R. Garafutdinov
    • 1
  1. 1.Institute of Biochemistry and Genetics, Ufa Science CentreRussian Academy of SciencesUfaRussia

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