期刊
NATURE METHODS
卷 8, 期 7, 页码 575-U84出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.1629
关键词
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资金
- US National Institutes of Health National Human Genome Research Institute [HG005097-01, 1RC2HG005613-01]
- Damon Runyon Cancer Research Foundation [DRG-2000 09]
- National Science Foundation [ECS-0335765]
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 similar to 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.
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