1. Droplet-based pyrosequencing using digital microfluidics.
- Author
-
Boles DJ, Benton JL, Siew GJ, Levy MH, Thwar PK, Sandahl MA, Rouse JL, Perkins LC, Sudarsan AP, Jalili R, Pamula VK, Srinivasan V, Fair RB, Griffin PB, Eckhardt AE, and Pollack MG
- Subjects
- Base Sequence, Candida genetics, Deoxyribonucleotides analysis, Deoxyribonucleotides genetics, Deoxyribonucleotides metabolism, Electrodes, Enzymes chemistry, Enzymes metabolism, Microfluidic Analytical Techniques instrumentation, Sequence Analysis, DNA instrumentation, Templates, Genetic, DNA, Fungal analysis, DNA, Fungal genetics, Microfluidic Analytical Techniques methods, Sequence Analysis, DNA methods
- Abstract
The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp Candida parapsilosis template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the C. parapsilosis sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., "sample-to-sequence" capability) could eventually be achieved using this low-cost platform.
- Published
- 2011
- Full Text
- View/download PDF