A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis.
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(R) 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.
Turcatti
Gerardo
G
Manteia Predictive Medicine S.A., Zone Industrielle, Coinsins, CH-1267, Switzerland. gerardo.turcatti@epfl.ch
Romieu
Anthony
A
Fedurco
Milan
M
Tairi
Ana-Paula
AP
D003854:Deoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000145:classification; D005456:Fluorescent Dyes / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D009930:Organic Chemicals / Q000737:chemistry; D017422:Sequence Analysis, DNA
C417664:Alexa594; D003854:Deoxyribonucleotides; D005456:Fluorescent Dyes; D009930:Organic Chemicals