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Ligation-free isothermal nucleic acid amplification

Authors
 Jeong Moon  ;  Jayeon Song  ;  Hyowon Jang  ;  Hyunju Kang  ;  Yong-Min Huh  ;  Hye Young Son  ;  Hyun Wook Rho  ;  Mirae Park  ;  Chandana S Talwar  ;  Kwang-Hyun Park  ;  Euijeon Woo  ;  Jaewoo Lim  ;  Eun-Kyung Lim  ;  Juyeon Jung  ;  Yongwon Jung  ;  Hyun Gyu Park  ;  Taejoon Kang 
Citation
 BIOSENSORS & BIOELECTRONICS, Vol.209(1) : 114256, 2022-08 
Journal Title
BIOSENSORS & BIOELECTRONICS
ISSN
 0956-5663 
Issue Date
2022-08
MeSH
Animals ; Biosensing Techniques* / methods ; CRISPR-Cas Systems* / genetics ; DNA / genetics ; Mice ; Nucleic Acid Amplification Techniques / methods ; RNA ; RNA, Messenger
Keywords
Cancer diagnosis ; Isothermal amplification ; Ligation-free ; Nucleic acid ; Phosphorothioate probe
Abstract
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.
Files in This Item:
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DOI
10.1016/j.bios.2022.114256
Appears in Collections:
1. College of Medicine (의과대학) > BioMedical Science Institute (의생명과학부) > 1. Journal Papers
1. College of Medicine (의과대학) > Dept. of Radiology (영상의학교실) > 1. Journal Papers
Yonsei Authors
Son, Hye Yeong(손혜영) ORCID logo https://orcid.org/0000-0001-5977-6784
Huh, Yong Min(허용민) ORCID logo https://orcid.org/0000-0002-9831-4475
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/189272
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