Fluorogenic Pyrosequencing in Microreactors

Abstract

High-throughput sequencing techniques are revolutionizing biology and promise to have a significant impact on the future of medicine. We have developed a scalable, parallel, low-cost method for nucleic acid sequencing that combines resealable microreactors and terminal-phosphate labeled fluorogenic nucleotides. To accomplish this, we have fabricated arrays of microreactors in polydimethylsiloxane (PDMS) that can be reversibly sealed, and have developed techniques for immobilizing different populations of identical DNA fragments specifically and efficiently within these microreactors. We have synthesized a series of terminal-phosphate labeled fluorogenic substrates for DNA polymerase which, upon their incorporation into a primer strand of DNA and subsequent digestion by a phosphatase, generate fluorescent dye molecules that are effectively trapped in the microreactors. By sequentially interrogating the microreactor array with solutions containing different fluorogenic nucleotide substrates, and recording which microreactors generate fluorescence, we can sequence the immobilized DNA. This “fluorogenic pyrosequencing” method promises significant advantages over existing sequencing technologies, and has the potential to be elegantly interfaced with the wide variety of PDMS-based microfluidic devices suitable for the preparation and amplification of DNA or RNA for sequencing, opening the possibility of one-chip, sample-to-sequence capabilities.