Ligation-free isothermal nucleic acid amplification

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In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.
Publisher
ELSEVIER ADVANCED TECHNOLOGY
Issue Date
2022-08
Language
English
Article Type
Article
Citation

BIOSENSORS & BIOELECTRONICS, v.209, pp.114256

ISSN
0956-5663
DOI
10.1016/j.bios.2022.114256
URI
http://hdl.handle.net/10203/296720
Appears in Collection
CH-Journal Papers(저널논문)CBE-Journal Papers(저널논문)
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