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Fluoride‐Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension
Author(s) -
Knapp Diana C.,
Serva Saulius,
D'Onofrio Jennifer,
Keller Angelika,
Lubys Arvydas,
Kurg Ants,
Remm Maido,
Engels Joachim W.
Publication year - 2011
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201001952
Subject(s) - linker , moiety , oligonucleotide , fluoride , nucleotide , fluorophore , combinatorial chemistry , chemistry , dna , dna synthesis , oligonucleotide synthesis , primer (cosmetics) , dna polymerase , reverse transcriptase , primer extension , stereochemistry , fluorescence , biophysics , biochemistry , polymerase chain reaction , biology , organic chemistry , gene , computer science , inorganic chemistry , physics , quantum mechanics , operating system
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.