Synthesis of 3'-O-Allyl Fluorescent Nucleotides as a Reversible terminator for Next Generation DNA Sequencing By Synthesis (SBS)
- Synthesis of 3'-O-Allyl Fluorescent Nucleotides as a Reversible terminator for Next Generation DNA Sequencing By Synthesis (SBS)
- 최승호; 이정석; 신동윤; 김종승
- Sequencing By Synthesis; Nucleotide; Reversible terminator
- Issue Date
- 106th Korea Chemical Society Meeting
- Next-Generation DNA Sequencing (NGS) opened DNA sequencing by synthesis (SBS) is one of the successful technology among Next-Generation DNA Sequencing (NGS). Current SBS technology is based on the CRT method, which is comprised of a three-step process of incorporating modified nucleotides, fluorescence imaging, and deprotecting for running the next cycle. This method requires dual-modified reversible terminators (DRTs), modified nucleotides having a reversible blocker on 3’-OH and fluorescent groups labeled on nucleobases for terminating DNA synthesis and for base-calling, respectively.
In this regard, we designed and synthesized mono-modified reversible terminators (MRTs) in which the reversible blocking group on 3’-OH plays a role as a fluorescence-signal reporter as well as a reversible terminator. Allyl-based MRT linker was prepared from 1,5-pentanediol in ## steps, in which Fmoc protected amine group are adopted to introduce fluorophores. Alkylation of linker with 3'-OH of deoxyribose, and triphosphorylation gave new SBS sequencing probes with mono-modified reversible terminators, which were successfully incorporated by an engineered DNA polymerase.
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