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Fluorogenic DNA sequencing in PDMS microreactors

SprMorn 添加于 2011/6/22 10:37:27  3029次阅读 | 0次推荐 | 0个评论

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.

作 者:Peter A Sims; William J Greenleaf; Haifeng Duan; X Sunney Xie
期刊名称: Nature Methods
期卷页: 2011-06-12 第卷 第期 ~页
学科领域:生命科学 » 生物物理、生物化学与分子生物学 » 生物物理、生物化学与分子生物学研究的新方法与新技术
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原文链接:http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.1629.html
DOI: doi:10.1038/nmeth.1629
ISBN: 1548-7091
关键词: Sequencing, Lab-on-a-chip, Microscopy, Genetics
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