Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method

Abstract The growing demand for cost-effective nucleic acid detection assays leads to an increasing number of different isothermal amplification reaction methods. However, all of the most efficient methods suffer from highly complex assay conditions due to the use of complicated primer sets and/or a...

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Autores principales:	Jens Fischbach, Marcus Frohme, Jörn Glökler
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/2f1a302be147416da7c0ce81a33cd8ae
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spelling	oai:doaj.org-article:2f1a302be147416da7c0ce81a33cd8ae2021-12-02T11:52:32ZHinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method10.1038/s41598-017-08067-x2045-2322https://doaj.org/article/2f1a302be147416da7c0ce81a33cd8ae2017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-08067-xhttps://doaj.org/toc/2045-2322Abstract The growing demand for cost-effective nucleic acid detection assays leads to an increasing number of different isothermal amplification reaction methods. However, all of the most efficient methods suffer from highly complex assay conditions due to the use of complicated primer sets and/or auxiliary enzymes. The present study describes the application of a new linker moiety that can be incorporated between a primer and a secondary target binding site which can act both as a block to polymerase extension as well as a hinge for refolding. This novel “hinge-primer” approach results in an efficient regeneration of the primer binding site and thus improves the strand-displacement and amplification process under isothermal conditions. Our investigations revealed that the reaction with forward and reverse hinge-primer including an abasic site is very efficient. The assay complexity can be reduced by combining the hinge-primer with a corresponding linear primer. Furthermore, the reaction speed can be increased by reducing the length of the amplified target sequence. We tested the sensitivity down to 104 copies and found a linear correlation between reaction time and input copy number. Our approach overcomes the usually cumbersome primer-design and extends the range of isothermal amplification methods using a polymerase with strand-displacement activity.Jens FischbachMarcus FrohmeJörn GlöklerNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jens Fischbach Marcus Frohme Jörn Glökler Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method
description	Abstract The growing demand for cost-effective nucleic acid detection assays leads to an increasing number of different isothermal amplification reaction methods. However, all of the most efficient methods suffer from highly complex assay conditions due to the use of complicated primer sets and/or auxiliary enzymes. The present study describes the application of a new linker moiety that can be incorporated between a primer and a secondary target binding site which can act both as a block to polymerase extension as well as a hinge for refolding. This novel “hinge-primer” approach results in an efficient regeneration of the primer binding site and thus improves the strand-displacement and amplification process under isothermal conditions. Our investigations revealed that the reaction with forward and reverse hinge-primer including an abasic site is very efficient. The assay complexity can be reduced by combining the hinge-primer with a corresponding linear primer. Furthermore, the reaction speed can be increased by reducing the length of the amplified target sequence. We tested the sensitivity down to 104 copies and found a linear correlation between reaction time and input copy number. Our approach overcomes the usually cumbersome primer-design and extends the range of isothermal amplification methods using a polymerase with strand-displacement activity.
format	article
author	Jens Fischbach Marcus Frohme Jörn Glökler
author_facet	Jens Fischbach Marcus Frohme Jörn Glökler
author_sort	Jens Fischbach
title	Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method
title_short	Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method
title_full	Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method
title_fullStr	Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method
title_full_unstemmed	Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method
title_sort	hinge-initiated primer-dependent amplification of nucleic acids (hip) – a new versatile isothermal amplification method
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/2f1a302be147416da7c0ce81a33cd8ae
work_keys_str_mv	AT jensfischbach hingeinitiatedprimerdependentamplificationofnucleicacidshipanewversatileisothermalamplificationmethod AT marcusfrohme hingeinitiatedprimerdependentamplificationofnucleicacidshipanewversatileisothermalamplificationmethod AT jornglokler hingeinitiatedprimerdependentamplificationofnucleicacidshipanewversatileisothermalamplificationmethod
_version_	1718394973689217024

Hinge-initiated Primer-dependent Amplification of Nucleic Acids (HIP) – A New Versatile Isothermal Amplification Method

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