Title: Fluorogenic DNA Sequencing in PDMS Microreactors

Speaker：Dr. Peter Sims
Department of Chemistry and Chemical Biology，Harvard University

Time: April, 29th, 2011, 10:00 AM
Location: BIOPIC No. 208, Conference Room 

ABSTRACT

Massively parallel sequencing has emerged as a powerful analytical tool with the potential to transform medicine. We have developed a multiplex sequencing-by-synthesis method combining terminal-phosphate labeled fluorogenic nucleotides (TPLFNs) and resealable microreactors. In the presence of phosphatase, the incorporation of a non-fluorescent TPLFN into a primer strand of DNA by DNA polymerase results in the generation of a fluorophore. We immobilize DNA templates within polydimethylsiloxane (PDMS) microreactors, sequentially introduce one of the four species of TPLFN, seal the microreactors, allow template-directed TPLFN incorporation, and measure the fluorescence from each chamber. This workflow allows DNA sequencing in a manner akin to pyrosequencing but with a fluorescence signal. “Fluorogenic pyrosequencing” combines benefits of pyrosequencing, such as rapid turn-around times, generation of native DNA, and single-color detection, with benefits of fluorescence-based approaches, such as highly sensitive detection and simple parallelization.