title: Fluorogenic sequencing using halogen-fluorescein-labeled nucleotides.
abstract: 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.
authors.affiliation: College of Engineering, Peking University, Beijing 100871 (China) http://www.gene.pku.edu.cn.|Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871 (China).
authors.forename: Zitian
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871 (China). hduan@pku.edu.cn.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Duan
authors.affiliation: Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871 (China).
authors.forename: Shuo
authors.initials: S
authors.lastname: Qiao
authors.affiliation: Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871 (China).
authors.forename: Wenxiong
authors.initials: W
authors.lastname: Zhou
authors.affiliation: Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871 (China).
authors.forename: Haiwei
authors.initials: H
authors.lastname: Qiu
authors.affiliation: Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871 (China).
authors.forename: Li
authors.initials: L
authors.lastname: Kang
authors.affiliation: Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871 (China).|Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138 (USA).
authors.forename: X Sunney
authors.initials: XS
authors.lastname: Xie
authors.affiliation: College of Engineering, Peking University, Beijing 100871 (China) http://www.gene.pku.edu.cn. yanyi@pku.edu.cn.|Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing 100871 (China). yanyi@pku.edu.cn.
authors.forename: Yanyi
authors.initials: Y
authors.lastname: Huang
mesh_terms: D004247:DNA / Q000737:chemistry / Q000235:genetics; D019793:Fluorescein / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006219:Halogens / Q000737:chemistry*; D007700:Kinetics; D009711:Nucleotides / Q000737:chemistry* / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling
chemical_list: D005456:Fluorescent Dyes; D006219:Halogens; D009711:Nucleotides; D004247:DNA; D019793:Fluorescein
keywords: DNA; fluorescent probes; fluorogenic sequencing; phosphate-labeled nucleotides; sequencer; sequencing by synthesis