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Fluoride-Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension

Fluorescent 2′-deoxynucleotides containing a protecting group at the 3′-O-position are reversible terminators that enable array-based DNA sequencing-by-synthesis (SBS) approaches. Herein, we describe the synthesis and full characterisation of four reversible terminators bearing a 3′-blocking moiety...

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Autores principales: Knapp, Diana C, Serva, Saulius, D'Onofrio, Jennifer, Keller, Angelika, Lubys, Arvydas, Kurg, Ants, Remm, Maido, Engels, Joachim W
Formato: Online Artículo Texto
Lenguaje:English
Publicado: WILEY-VCH Verlag 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3110862/
https://www.ncbi.nlm.nih.gov/pubmed/21294195
http://dx.doi.org/10.1002/chem.201001952
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author Knapp, Diana C
Serva, Saulius
D'Onofrio, Jennifer
Keller, Angelika
Lubys, Arvydas
Kurg, Ants
Remm, Maido
Engels, Joachim W
author_facet Knapp, Diana C
Serva, Saulius
D'Onofrio, Jennifer
Keller, Angelika
Lubys, Arvydas
Kurg, Ants
Remm, Maido
Engels, Joachim W
author_sort Knapp, Diana C
collection PubMed
description Fluorescent 2′-deoxynucleotides containing a protecting group at the 3′-O-position are reversible terminators that enable array-based DNA sequencing-by-synthesis (SBS) approaches. Herein, we describe the synthesis and full characterisation of four reversible terminators bearing a 3′-blocking moiety and a linker-dye system that is removable under the same fluoride-based treatment. Each nucleotide analogue has a different fluorophore attached to the base through a fluoride-cleavable linker and a 2-cyanoethyl moiety as the 3′-blocking group, which can be removed by using a fluoride treatment as well. Furthermore, we identified a DNA polymerase, namely, RevertAid M-MuLV reverse transcriptase, which can incorporate the four modified reversible terminators. The synthesised nucleotides and the optimised DNA polymerase were used on CodeLink slides spotted with hairpin oligonucleotides to demonstrate their potential in a cyclic reversible terminating approach.
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spelling pubmed-31108622011-06-14 Fluoride-Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension Knapp, Diana C Serva, Saulius D'Onofrio, Jennifer Keller, Angelika Lubys, Arvydas Kurg, Ants Remm, Maido Engels, Joachim W Chemistry Full Paper Fluorescent 2′-deoxynucleotides containing a protecting group at the 3′-O-position are reversible terminators that enable array-based DNA sequencing-by-synthesis (SBS) approaches. Herein, we describe the synthesis and full characterisation of four reversible terminators bearing a 3′-blocking moiety and a linker-dye system that is removable under the same fluoride-based treatment. Each nucleotide analogue has a different fluorophore attached to the base through a fluoride-cleavable linker and a 2-cyanoethyl moiety as the 3′-blocking group, which can be removed by using a fluoride treatment as well. Furthermore, we identified a DNA polymerase, namely, RevertAid M-MuLV reverse transcriptase, which can incorporate the four modified reversible terminators. The synthesised nucleotides and the optimised DNA polymerase were used on CodeLink slides spotted with hairpin oligonucleotides to demonstrate their potential in a cyclic reversible terminating approach. WILEY-VCH Verlag 2011-03-01 2011-02-03 /pmc/articles/PMC3110862/ /pubmed/21294195 http://dx.doi.org/10.1002/chem.201001952 Text en Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://creativecommons.org/licenses/by/2.5/ Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.
spellingShingle Full Paper
Knapp, Diana C
Serva, Saulius
D'Onofrio, Jennifer
Keller, Angelika
Lubys, Arvydas
Kurg, Ants
Remm, Maido
Engels, Joachim W
Fluoride-Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension
title Fluoride-Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension
title_full Fluoride-Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension
title_fullStr Fluoride-Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension
title_full_unstemmed Fluoride-Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension
title_short Fluoride-Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension
title_sort fluoride-cleavable, fluorescently labelled reversible terminators: synthesis and use in primer extension
topic Full Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3110862/
https://www.ncbi.nlm.nih.gov/pubmed/21294195
http://dx.doi.org/10.1002/chem.201001952
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