Since the first human genome determination a decade ago, the cost of genome sequencing has been reducing with 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 demonstratesequencing with ~99% raw accuracy. Our ‘fluorogenicpyrosequencing’ 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.

 

 

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).

 

A compelling new area of genome research is single cell sequencing. To determine the genome of a given cell is in fact a single molecule experiment. We are developing methodologies for single cell genome and transcriptome analyses., and exploring the variety of applications.