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Chemistry. 2011 Mar 1;17(10):2903-15. doi: 10.1002/chem.201001952. Epub 2011 Feb 3.

Fluoride-cleavable, fluorescently labelled reversible terminators: synthesis and use in primer extension.

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  • 1Institut für Organische Chemie und Chemische Biologie, J.W. Goethe Universität Frankfurt am Main, Max-von-Laue Strasse 7, 60438 Frankfurt am Main, Germany.

Abstract

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.

Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PMID:
21294195
[PubMed - indexed for MEDLINE]
PMCID:
PMC3110862
Free PMC Article
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