Nucleic Acids Research Advance Access published online on February 7, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn021
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A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis
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
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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