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Nucleic Acids Research Advance Access originally published online on February 7, 2008
Nucleic Acids Research 2008 36(4):e25; doi:10.1093/nar/gkn021
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Nucleic Acids Research, 2008, Vol. 36, No. 4 e25
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis{dagger}

Gerardo Turcatti*, Anthony Romieu, Milan Fedurco and Ana-Paula Tairi

Manteia Predictive Medicine S.A., Zone Industrielle, Coinsins, CH-1267, Switzerland

*To whom correspondence should be addressed. Tel: +41 21 693 9666; Fax: +41 21 693 9667; Email: gerardo.turcatti{at}epfl.ch

Received September 4, 2007. Revised January 15, 2008. Accepted January 15, 2008.

Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators enabling array-based DNA sequencing by synthesis (SBS) approaches. Herein, we describe the synthesis of a new family of 3'-OH unprotected cleavable fluorescent 2'-deoxynucleotides and their evaluation as reversible terminators for high-throughput DNA SBS strategies. In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor® 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base. Their use in SBS leaded to a signal–no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out). Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA templates.

Present address: Gerardo Turcatti, EPFL, School of Life Sciences, AAB013, Station 15, CH-1015 Lausanne, Switzerland

{dagger}This work is based on experiments done at Manteia Predictive Medicine SA, a Swiss-based private company developing technologies for high-throughput DNA sequencing, jointly acquired by Lynx Therapeutics Inc. and Solexa Ltd. Both companies completed a merger in March 2005 and became Solexa Inc. In January 2007, Solexa was acquired by Illumina Inc. (www.illumina.com).


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