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Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction

The exponential amplification reaction (EXPAR) is an emerging isothermal nucleic acid amplification method with high potential for molecular diagnostics due to its isothermal nature and high amplification efficiency. However, the use of EXPAR is limited by the high levels of non-specific amplificati...

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Autores principales: Mok, Ellie, Wee, Eugene, Wang, Yuling, Trau, Matt
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133538/
https://www.ncbi.nlm.nih.gov/pubmed/27910874
http://dx.doi.org/10.1038/srep37837
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author Mok, Ellie
Wee, Eugene
Wang, Yuling
Trau, Matt
author_facet Mok, Ellie
Wee, Eugene
Wang, Yuling
Trau, Matt
author_sort Mok, Ellie
collection PubMed
description The exponential amplification reaction (EXPAR) is an emerging isothermal nucleic acid amplification method with high potential for molecular diagnostics due to its isothermal nature and high amplification efficiency. However, the use of EXPAR is limited by the high levels of non-specific amplification. Hence, methods that can improve the specificity of EXPAR are desired to facilitate its widespread adoption in practice. Herein, we proposed a strategy to improve EXPAR performance by using molecular enhancers. Eight small molecules were investigated, including ethylene glycol, propylene glycol, betaine, dimethyl sulfoxide (DMSO), trehalose, tetramethylammonium chloride (TMAC), bovine serum albumin (BSA) and single-stranded binding (SSB) proteins. A combination of kinetic and end-point analysis was adopted to investigate how these molecules affected EXPAR performance. Trehalose, TMAC, BSA and SSB proteins were found to have positive effects on EXPAR with trehalose being able to increase the efficiency of EXPAR. In contrast, TMAC, BSA and SSB proteins were shown to increase the specificity of EXPAR. We applied our findings to demonstrate the combination of trehalose and TMAC could simultaneously improve both the efficiency and specificity of an EXPAR-based miRNA detection method. The information provided in this study may serve as a reference to benefit the wider isothermal amplification community.
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spelling pubmed-51335382017-01-27 Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction Mok, Ellie Wee, Eugene Wang, Yuling Trau, Matt Sci Rep Article The exponential amplification reaction (EXPAR) is an emerging isothermal nucleic acid amplification method with high potential for molecular diagnostics due to its isothermal nature and high amplification efficiency. However, the use of EXPAR is limited by the high levels of non-specific amplification. Hence, methods that can improve the specificity of EXPAR are desired to facilitate its widespread adoption in practice. Herein, we proposed a strategy to improve EXPAR performance by using molecular enhancers. Eight small molecules were investigated, including ethylene glycol, propylene glycol, betaine, dimethyl sulfoxide (DMSO), trehalose, tetramethylammonium chloride (TMAC), bovine serum albumin (BSA) and single-stranded binding (SSB) proteins. A combination of kinetic and end-point analysis was adopted to investigate how these molecules affected EXPAR performance. Trehalose, TMAC, BSA and SSB proteins were found to have positive effects on EXPAR with trehalose being able to increase the efficiency of EXPAR. In contrast, TMAC, BSA and SSB proteins were shown to increase the specificity of EXPAR. We applied our findings to demonstrate the combination of trehalose and TMAC could simultaneously improve both the efficiency and specificity of an EXPAR-based miRNA detection method. The information provided in this study may serve as a reference to benefit the wider isothermal amplification community. Nature Publishing Group 2016-12-02 /pmc/articles/PMC5133538/ /pubmed/27910874 http://dx.doi.org/10.1038/srep37837 Text en Copyright © 2016, The Author(s) http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Mok, Ellie
Wee, Eugene
Wang, Yuling
Trau, Matt
Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction
title Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction
title_full Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction
title_fullStr Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction
title_full_unstemmed Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction
title_short Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction
title_sort comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133538/
https://www.ncbi.nlm.nih.gov/pubmed/27910874
http://dx.doi.org/10.1038/srep37837
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