Fluorogenic DNA Sequencing

Since the first human genome determination a decade ago, the cost of genome sequencing has been reducing at a rate faster than the Moores’ law. Various faster and cheaper sequencing methods offer exciting possibilities for biology and medicine.

We have developed a multiplex sequencing-by-synthesis method combining terminal phosphate–labeled fluorogenic nucleotides (TPLFNs) and resealablepolydimethylsiloxane (PDMS) microreactors. In the presence of phosphatase, primer extension by DNA polymerase using nonfluorescentTPLFNs 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 sequencing with ~99% raw accuracy. Our ‘fluorogenicpyrosequencing’ offers the 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.

References

Sims, Peter A.; Greenleaf, William J.; Duan, Haifeng; Xie, X. Sunney.“Fluorogenic DNA Sequencing in PDMS Microreactors,” Nat Methods 8:575-580 (2011).

Steen, Jason A; Cooper, Matthew A. “Fluorogenic Pyrosequencing in Microreactors,” Nat Methods 8:548-549 (2011).