Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification

Abstract Herein, we describe a phosphorothioated hairpin-assisted isothermal amplification (PHAmp) method for detection of a target nucleic acid. The hairpin probe (HP) is designed to contain a 5′ phosphorothioate (PS)-modified overhang, a target recognition site, and a 3′ self-priming (SP) region....

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Autores principales:	Yujin Jung, Jayeon Song, Hyun Gyu Park
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e1a9cc547b4140e4b957d0a6bf23902e
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spelling	oai:doaj.org-article:e1a9cc547b4140e4b957d0a6bf23902e2021-12-02T15:51:13ZUltrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification10.1038/s41598-021-87948-82045-2322https://doaj.org/article/e1a9cc547b4140e4b957d0a6bf23902e2021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-87948-8https://doaj.org/toc/2045-2322Abstract Herein, we describe a phosphorothioated hairpin-assisted isothermal amplification (PHAmp) method for detection of a target nucleic acid. The hairpin probe (HP) is designed to contain a 5′ phosphorothioate (PS)-modified overhang, a target recognition site, and a 3′ self-priming (SP) region. Upon binding to the target nucleic acid, the HP opens and the SP region is rearranged to serve as a primer. The subsequent process of strand displacement DNA synthesis recycles the bound target to open another HP and produces an extended HP (EP) with a PS-DNA/DNA duplex at the end, which would be readily denatured due to its reduced thermal stability. The trigger then binds to the denatured 3′ end of the EP and is extended, producing an intermediate double-stranded (ds) DNA product (IP). The trigger also binds to the denatured 3′ end of the IP, and its extension produces the final dsDNA product along with concomitant displacement and recycling of EP. By monitoring the dsDNA products, the target nucleic acid can be identified down to 0.29 fM with a wide dynamic range from 1 nM to 1 fM yielding an excellent specificity to discriminate even a single base-mismatched target. The unique design principle could provide new insights into the development of novel isothermal amplification methods for nucleic acid detection.Yujin JungJayeon SongHyun Gyu ParkNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-8 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yujin Jung Jayeon Song Hyun Gyu Park Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification
description	Abstract Herein, we describe a phosphorothioated hairpin-assisted isothermal amplification (PHAmp) method for detection of a target nucleic acid. The hairpin probe (HP) is designed to contain a 5′ phosphorothioate (PS)-modified overhang, a target recognition site, and a 3′ self-priming (SP) region. Upon binding to the target nucleic acid, the HP opens and the SP region is rearranged to serve as a primer. The subsequent process of strand displacement DNA synthesis recycles the bound target to open another HP and produces an extended HP (EP) with a PS-DNA/DNA duplex at the end, which would be readily denatured due to its reduced thermal stability. The trigger then binds to the denatured 3′ end of the EP and is extended, producing an intermediate double-stranded (ds) DNA product (IP). The trigger also binds to the denatured 3′ end of the IP, and its extension produces the final dsDNA product along with concomitant displacement and recycling of EP. By monitoring the dsDNA products, the target nucleic acid can be identified down to 0.29 fM with a wide dynamic range from 1 nM to 1 fM yielding an excellent specificity to discriminate even a single base-mismatched target. The unique design principle could provide new insights into the development of novel isothermal amplification methods for nucleic acid detection.
format	article
author	Yujin Jung Jayeon Song Hyun Gyu Park
author_facet	Yujin Jung Jayeon Song Hyun Gyu Park
author_sort	Yujin Jung
title	Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification
title_short	Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification
title_full	Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification
title_fullStr	Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification
title_full_unstemmed	Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification
title_sort	ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/e1a9cc547b4140e4b957d0a6bf23902e
work_keys_str_mv	AT yujinjung ultrasensitivenucleicaciddetectionbasedonphosphorothioatedhairpinassistedisothermalamplification AT jayeonsong ultrasensitivenucleicaciddetectionbasedonphosphorothioatedhairpinassistedisothermalamplification AT hyungyupark ultrasensitivenucleicaciddetectionbasedonphosphorothioatedhairpinassistedisothermalamplification
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Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification

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