Fluorogenic Sequencing Using Halogen-Fluorescein-Labeled Nucleotides

Fluorogenic sequencing is a sequencing‐by‐synthesis technology that combines the advantages of pyrosequencing and fluorescence detection. With native duplex DNA as the major product, we employ polymerase to incorporate the complement‐ arily matched terminal phosphate‐labeled fluorogenic nucleotides into the DNA template and release halogen‐fluorescein as the reporter. This red‐emitting fluorophore successfully avoids spectral overlap with the autofluorescence background of the flow chip. We fully characterized the enzymatic reaction kinetics of the new substrates, and performed a 35‐base sequencing experiment with 60 reaction cycles. Our achievement expands the substrate repertoire for fluorogenic sequencing, and extends the spectral range to obtain better signal‐to‐background performance. We demonstrate fluorogenic sequencing of DNA with halogen‐tuned fluorescein‐based terminal‐phosphate‐labeled nucleotides. These fluorogenic substrates inherit the superior fluorescence properties of green‐emitting fluorescein, but with longer emission wavelength to improve signal‐to‐background ratio in sequencing.