Design and Synthesis of 3'-Oxygen-Modified Cleavable Nucleotide Reversible Terminators for Scarless DNA Sequencing by Synthesis
This dissertation describes the design and synthesis of novel cleavable fluorescent/anchor modified nucleotide reversible terminators using 3’-O-dithiomethyl (3’-O-DTM; 3’-O-SS) as a linker to directly or indirectly attach a fluorescent reporter to achieve scarless DNA Sequencing by Synthesis (SBS)....
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ndltd-columbia.edu-oai-academiccommons.columbia.edu-10.7916-D8XD2HST2019-05-09T15:15:48ZDesign and Synthesis of 3'-Oxygen-Modified Cleavable Nucleotide Reversible Terminators for Scarless DNA Sequencing by SynthesisHsieh, Min-Kang2018ThesesChemical engineeringBiochemistryBiomedical engineeringNucleotide sequenceThis dissertation describes the design and synthesis of novel cleavable fluorescent/anchor modified nucleotide reversible terminators using 3’-O-dithiomethyl (3’-O-DTM; 3’-O-SS) as a linker to directly or indirectly attach a fluorescent reporter to achieve scarless DNA Sequencing by Synthesis (SBS). To develop these nucleotide analogues for four-color SBS, two nucleotide analogues (3’-O-ROX-SS-dATP and 3’-O-BodipyFL-SS-dTTP) with directly attached fluorescent dyes and two other nucleotide analogues with directly attached biotin or trans-cyclooctene (TCO) as anchors (3’-O-Biotin-SS-dCTP and 3’-O-TCO-SS-dGTP) were successfully designed and synthesized. The nucleotide analogues with a PEG-elongated linker (3’-O-ROX-PEG4-SS-dATP, 3’-O-BodipyFL-PEG4-SS-dTTP, 3’-O-Biotin-PEG4-SS-dCTP and 3’-O-TCO-PEG4-SS-dGTP) were also designed and synthesized to optimize their incorporation efficiency in polymerase reactions. In our design, Biotin and TCO were demonstrated to be anchor moieties with high efficiency and specificity for binding with fluorescently labeled streptavidin and tetrazine, respectively. The DNA extension products produced by polymerase incorporation of 3’-O-Biotin-SS-dCTP and 3’-O-Biotin-PEG4-SS-dCTP were accurately identified by binding to Cy5-labeled streptavidin, while the DNA extension products produced by polymerase incorporation of 3’-O-TCO-SS-dGTP and 3’-O-TCO-PEG4-SS-dGTP were identified with equal precision by reaction with TAMRA-labeled tetrazine. A proof-of-concept experiment was conducted to demonstrate four-color scarless SBS using the novel nucleotide analogues described above.Englishhttps://doi.org/10.7916/D8XD2HST |
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English |
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Chemical engineering Biochemistry Biomedical engineering Nucleotide sequence |
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Chemical engineering Biochemistry Biomedical engineering Nucleotide sequence Hsieh, Min-Kang Design and Synthesis of 3'-Oxygen-Modified Cleavable Nucleotide Reversible Terminators for Scarless DNA Sequencing by Synthesis |
description |
This dissertation describes the design and synthesis of novel cleavable fluorescent/anchor modified nucleotide reversible terminators using 3’-O-dithiomethyl (3’-O-DTM; 3’-O-SS) as a linker to directly or indirectly attach a fluorescent reporter to achieve scarless DNA Sequencing by Synthesis (SBS). To develop these nucleotide analogues for four-color SBS, two nucleotide analogues (3’-O-ROX-SS-dATP and 3’-O-BodipyFL-SS-dTTP) with directly attached fluorescent dyes and two other nucleotide analogues with directly attached biotin or trans-cyclooctene (TCO) as anchors (3’-O-Biotin-SS-dCTP and 3’-O-TCO-SS-dGTP) were successfully designed and synthesized. The nucleotide analogues with a PEG-elongated linker (3’-O-ROX-PEG4-SS-dATP, 3’-O-BodipyFL-PEG4-SS-dTTP, 3’-O-Biotin-PEG4-SS-dCTP and 3’-O-TCO-PEG4-SS-dGTP) were also designed and synthesized to optimize their incorporation efficiency in polymerase reactions. In our design, Biotin and TCO were demonstrated to be anchor moieties with high efficiency and specificity for binding with fluorescently labeled streptavidin and tetrazine, respectively. The DNA extension products produced by polymerase incorporation of 3’-O-Biotin-SS-dCTP and 3’-O-Biotin-PEG4-SS-dCTP were accurately identified by binding to Cy5-labeled streptavidin, while the DNA extension products produced by polymerase incorporation of 3’-O-TCO-SS-dGTP and 3’-O-TCO-PEG4-SS-dGTP were identified with equal precision by reaction with TAMRA-labeled tetrazine. A proof-of-concept experiment was conducted to demonstrate four-color scarless SBS using the novel nucleotide analogues described above. |
author |
Hsieh, Min-Kang |
author_facet |
Hsieh, Min-Kang |
author_sort |
Hsieh, Min-Kang |
title |
Design and Synthesis of 3'-Oxygen-Modified Cleavable Nucleotide Reversible Terminators for Scarless DNA Sequencing by Synthesis |
title_short |
Design and Synthesis of 3'-Oxygen-Modified Cleavable Nucleotide Reversible Terminators for Scarless DNA Sequencing by Synthesis |
title_full |
Design and Synthesis of 3'-Oxygen-Modified Cleavable Nucleotide Reversible Terminators for Scarless DNA Sequencing by Synthesis |
title_fullStr |
Design and Synthesis of 3'-Oxygen-Modified Cleavable Nucleotide Reversible Terminators for Scarless DNA Sequencing by Synthesis |
title_full_unstemmed |
Design and Synthesis of 3'-Oxygen-Modified Cleavable Nucleotide Reversible Terminators for Scarless DNA Sequencing by Synthesis |
title_sort |
design and synthesis of 3'-oxygen-modified cleavable nucleotide reversible terminators for scarless dna sequencing by synthesis |
publishDate |
2018 |
url |
https://doi.org/10.7916/D8XD2HST |
work_keys_str_mv |
AT hsiehminkang designandsynthesisof3oxygenmodifiedcleavablenucleotidereversibleterminatorsforscarlessdnasequencingbysynthesis |
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1719047038895652864 |