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Fluorogenic Sequencing Using Halogen‐Fluorescein‐Labeled Nucleotides
Author(s) -
Chen Zitian,
Duan Haifeng,
Qiao Shuo,
Zhou Wenxiong,
Qiu Haiwei,
Kang Li,
Xie X. Sunney,
Huang Yanyi
Publication year - 2015
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201500117
Subject(s) - dna nanoball sequencing , fluorophore , pyrosequencing , nucleotide , single molecule real time sequencing , multiplex , chemistry , fluorescein , dna , fluorescence , dna sequencing , combinatorial chemistry , autofluorescence , biochemistry , microbiology and biotechnology , biology , dna sequencer , base sequence , gene , genomic library , genetics , physics , quantum mechanics
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.