Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators |
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Authors: | Ju Jingyue Kim Dae Hyun Bi Lanrong Meng Qinglin Bai Xiaopeng Li Zengmin Li Xiaoxu Marma Mong Sano Shi Shundi Wu Jian Edwards John R Romu Aireen Turro Nicholas J |
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Institution: | Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA. ju@c2b2.columbia.edu |
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Abstract: | DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to decipher DNA sequences. We report here the construction of such a DNA sequencing system using molecular engineering approaches. In this approach, four nucleotides (A, C, G, T) are modified as reversible terminators by attaching a cleavable fluorophore to the base and capping the 3'-OH group with a small chemically reversible moiety so that they are still recognized by DNA polymerase as substrates. We found that an allyl moiety can be used successfully as a linker to tether a fluorophore to 3'-O-allyl-modified nucleotides, forming chemically cleavable fluorescent nucleotide reversible terminators, 3'-O-allyl-dNTPs-allyl-fluorophore, for application in SBS. The fluorophore and the 3'-O-allyl group on a DNA extension product, which is generated by incorporating 3'-O-allyl-dNTPs-allyl-fluorophore in a polymerase reaction, are removed simultaneously in 30 s by Pd-catalyzed deallylation in aqueous buffer solution. This one-step dual-deallylation reaction thus allows the reinitiation of the polymerase reaction and increases the SBS efficiency. DNA templates consisting of homopolymer regions were accurately sequenced by using this class of fluorescent nucleotide analogues on a DNA chip and a four-color fluorescent scanner. |
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