Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

Jingyue Ju, Dae Hyun Kim, Lanrong Bi, Qinglin Meng, Xiaopeng Bai, Zengmin Li, Xiaoxu Li, Mong Sano Marma, Shundi Shi, Jian Wu, John R. Edwards, Aireen Romu, Nicholas J. Turro

Research output: Contribution to journalArticlepeer-review

163 Scopus citations

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.

Original languageEnglish
Pages (from-to)19635-19640
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume103
Issue number52
DOIs
StatePublished - Dec 26 2006

Keywords

  • DNA chip

Fingerprint

Dive into the research topics of 'Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators'. Together they form a unique fingerprint.

Cite this