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

doi:10.1073/pnas.0609513103

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

*Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032; and
Departments of ^†Chemical Engineering,
^¶Chemistry, and
^§Biomedical Engineering, Columbia University, New York, NY 10027

Contributed by Nicholas J. Turro, October 26, 2006 (received for review October 1, 2006)

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.

DNA chip

Footnotes

^‡To whom correspondence may be addressed at:
Room 405A, Russ Berrie Medical Science Pavilion, Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032.
E-mail: ju{at}c2b2.columbia.edu or njt3{at}columbia.edu
Author contributions: J.J. and N.J.T. designed research; D.H.K., L.B., Q.M., X.B., Z.L., X.L., M.S.M., S.S., J.W., J.R.E., and A.R. performed research; and J.J., D.H.K., and Q.M. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0609513103/DC1.
Abbreviations:

SBS,

sequencing by synthesis;

bodipy,

4,4-difluoro-4-bora-3α,4α-diaza-s-indacene;

ROX,

6-carboxy-X-rhodamine;

R6G,

6-carboxyrhodamine-6G.
Freely available online through the PNAS open access option.