title: Fluorogenic DNA sequencing in PDMS microreactors.
abstract: We developed a multiplex sequencing-by-synthesis method combining terminal phosphate-labeled fluorogenic nucleotides (TPLFNs) and resealable polydimethylsiloxane (PDMS) microreactors. In the presence of phosphatase, primer extension by DNA polymerase using nonfluorescent TPLFNs generates fluorophores, which are confined in the microreactors and detected. We immobilized primed DNA templates in the microreactors, then sequentially introduced one of the four identically labeled TPLFNs, sealed the microreactors and recorded a fluorescence image after template-directed primer extension. With cycle times of <10 min, we demonstrate 30 base reads with ∼99% raw accuracy. Our 'fluorogenic pyrosequencing' offers benefits of pyrosequencing, such as rapid turnaround, one-color detection and generation of native DNA, along with high detection sensitivity and simplicity of parallelization because simultaneous real-time monitoring of all microreactors is not required.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Sims
authors.forename: William J
authors.initials: WJ
authors.lastname: Greenleaf
authors.forename: Haifeng
authors.initials: H
authors.lastname: Duan
authors.forename: X Sunney
authors.initials: XS
authors.lastname: Xie
mesh_terms: D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D004129:Dimethylpolysiloxanes / Q000737:chemistry*; D005453:Fluorescence; D008832:Microchemistry / Q000295:instrumentation; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D011108:Polymers / Q000737:chemistry*; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*
chemical_list: D004129:Dimethylpolysiloxanes; D011108:Polymers; D004247:DNA