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

Synthesis of circular DNA templates with T4 RNA ligase for rolling circle amplification 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Biology Springer Journals

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

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Publisher
Springer Journals
Copyright
Copyright © 2017 by Pleiades Publishing, Inc.
Subject
Life Sciences; Life Sciences, general; Biochemistry, general; Human Genetics
ISSN
0026-8933
eISSN
1608-3245
D.O.I.
10.1134/S0026893317040161
Publisher site
See Article on Publisher Site

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.

Journal

Molecular BiologySpringer Journals

Published: Aug 23, 2017

References

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