Design and synthesis of a chemically cleavable fluorescent nucleotide, 3'-O-allyl-dGTP-allyl-bodipy-FL-510, as a reversible terminator for DNA sequencing by synthesis.

We report the construction of a novel chemically cleavable fluorescent labeling system based on an allyl group to modify nucleotide for DNA sequencing by synthesis (SBS). We have found that an allyl moiety can be used successfully as a linker to tether a fluorophore to a 3'-O-allyl-modified nucleotide, forming a chemically cleavable reversible terminator, 3'-O-allyl-dGTP-allyl-Bodipy-FL-510, for application in SBS. The fluorophore and the 3'-allyl group on a DNA extension product, which is generated by incorporating 3'-O-allyl-dGTP-allyl-Bodipy-FL-510, are removed simultaneously in 30 s by Pd-catalyzed deallylation in aqueous buffer solution. The resulting DNA product is successfully used as a primer for further extension in polymerase reaction. This one-pot dual-deallylation reaction thus allows the reinitiation of the polymerase reaction and increases the SBS efficiency.