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.
journal_name
Nat Methodsjournal_title
Nature methodsauthors
Sims PA,Greenleaf WJ,Duan H,Xie XSdoi
10.1038/nmeth.1629subject
Has Abstractpub_date
2011-06-12 00:00:00pages
575-80issue
7eissn
1548-7091issn
1548-7105pii
nmeth.1629journal_volume
8pub_type
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