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High-Efficiency Reverse (5′→3′) Synthesis of Complex DNA Microarrays

DNA microarrays are important analytical tools in genetics and have recently found multiple new biotechnological roles in applications requiring free 3′ terminal hydroxyl groups, particularly as a starting point for enzymatic extension via DNA or RNA polymerases. Here we demonstrate the highly effic...

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Autores principales: Hölz, Kathrin, Hoi, Julia K., Schaudy, Erika, Somoza, Veronika, Lietard, Jory, Somoza, Mark M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180089/
https://www.ncbi.nlm.nih.gov/pubmed/30305718
http://dx.doi.org/10.1038/s41598-018-33311-3
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author Hölz, Kathrin
Hoi, Julia K.
Schaudy, Erika
Somoza, Veronika
Lietard, Jory
Somoza, Mark M.
author_facet Hölz, Kathrin
Hoi, Julia K.
Schaudy, Erika
Somoza, Veronika
Lietard, Jory
Somoza, Mark M.
author_sort Hölz, Kathrin
collection PubMed
description DNA microarrays are important analytical tools in genetics and have recently found multiple new biotechnological roles in applications requiring free 3′ terminal hydroxyl groups, particularly as a starting point for enzymatic extension via DNA or RNA polymerases. Here we demonstrate the highly efficient reverse synthesis of complex DNA arrays using a photolithographic approach. The method is analogous to conventional solid phase synthesis but makes use of phosphoramidites with the benzoyl-2-(2-nitrophenyl)-propoxycarbonyl (BzNPPOC) photolabile protecting group on the 3′-hydroxyl group. The use of BzNPPOC, with more than twice the photolytic efficiency of the 2-(2-nitrophenyl)-propoxycarbonyl (NPPOC) previously used for 5′→3′ synthesis, combined with additional optimizations to the coupling and oxidation reactions results in an approximately 3-fold improvement in the reverse synthesis efficiency of complex arrays of DNA oligonucleotides. The coupling efficiencies of the reverse phosphoramidites are as good as those of regular phosphoramidites, resulting in comparable yields. Microarrays of DNA surface tethered on the 5′ end and with free 3′ hydroxyl termini can be synthesized quickly and with similarly high stepwise coupling efficiency as microarrays using conventional 3′→5′ synthesis.
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spelling pubmed-61800892018-10-15 High-Efficiency Reverse (5′→3′) Synthesis of Complex DNA Microarrays Hölz, Kathrin Hoi, Julia K. Schaudy, Erika Somoza, Veronika Lietard, Jory Somoza, Mark M. Sci Rep Article DNA microarrays are important analytical tools in genetics and have recently found multiple new biotechnological roles in applications requiring free 3′ terminal hydroxyl groups, particularly as a starting point for enzymatic extension via DNA or RNA polymerases. Here we demonstrate the highly efficient reverse synthesis of complex DNA arrays using a photolithographic approach. The method is analogous to conventional solid phase synthesis but makes use of phosphoramidites with the benzoyl-2-(2-nitrophenyl)-propoxycarbonyl (BzNPPOC) photolabile protecting group on the 3′-hydroxyl group. The use of BzNPPOC, with more than twice the photolytic efficiency of the 2-(2-nitrophenyl)-propoxycarbonyl (NPPOC) previously used for 5′→3′ synthesis, combined with additional optimizations to the coupling and oxidation reactions results in an approximately 3-fold improvement in the reverse synthesis efficiency of complex arrays of DNA oligonucleotides. The coupling efficiencies of the reverse phosphoramidites are as good as those of regular phosphoramidites, resulting in comparable yields. Microarrays of DNA surface tethered on the 5′ end and with free 3′ hydroxyl termini can be synthesized quickly and with similarly high stepwise coupling efficiency as microarrays using conventional 3′→5′ synthesis. Nature Publishing Group UK 2018-10-10 /pmc/articles/PMC6180089/ /pubmed/30305718 http://dx.doi.org/10.1038/s41598-018-33311-3 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Hölz, Kathrin
Hoi, Julia K.
Schaudy, Erika
Somoza, Veronika
Lietard, Jory
Somoza, Mark M.
High-Efficiency Reverse (5′→3′) Synthesis of Complex DNA Microarrays
title High-Efficiency Reverse (5′→3′) Synthesis of Complex DNA Microarrays
title_full High-Efficiency Reverse (5′→3′) Synthesis of Complex DNA Microarrays
title_fullStr High-Efficiency Reverse (5′→3′) Synthesis of Complex DNA Microarrays
title_full_unstemmed High-Efficiency Reverse (5′→3′) Synthesis of Complex DNA Microarrays
title_short High-Efficiency Reverse (5′→3′) Synthesis of Complex DNA Microarrays
title_sort high-efficiency reverse (5′→3′) synthesis of complex dna microarrays
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180089/
https://www.ncbi.nlm.nih.gov/pubmed/30305718
http://dx.doi.org/10.1038/s41598-018-33311-3
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