[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Next-generation sequencing (NGS) technologies recently developed are now used for study of genomes from various organisms. Sequencing-by-synthesis (SBS) is a key strategy in the NGS. The SBS uses nucleotides so-called dual-modified reversible terminators (DRTs) in which bases are labeled with fluorophores and 3'-OH is protected with a reversibly cleavable chemical group, respectively. In this study, we examined the possibility of performing SBS with mono-modified reversible terminators (MRTs), in which the reversible blocking group on the 3'-OH plays a dual role as a fluorescent signal report as well as a chemical protection. We studied cyclic reversible termination by using two MRTs (dA and dT), wherein the modifications were two different fluorophores and cleavable to regenerate a free 3'-OH. We here demonstrated that SBS could be achieved with incorporation of MRTs by a DNA polymerase and correct base-calls based on the two different colors from the fluorophores.
[Show abstract][Hide abstract] ABSTRACT: Oligonucleotides composed of natural nucleotides are inapplicable for biotechnical and therapeutic use due to its instability under biological conditions. Therminator DNA polymerases, mutant DNA polymerases of thermophilic marine archaea, show that they can efficiently synthesize fully 2'-fluoro-modified (2'F-) oligonucleotides. Furthermore, the sequence analysis reveals that the oligonucleotide sequence is highly accurate, especially the fidelity of a 2'F-oligonucleotide synthesized by Therminator II is more accurate than that of natural RNA synthesized by conventional RNA polymerase. These finding would be helpful for the synthesis of chemically modified oligonucleotides, for the use of biotechnical or medical applications.
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