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Droplet-Based Pyrosequencing Using Digital Microfluidics.
Anal Chem. 2011 Sep 20;
Authors: Boles DJ, Benton JL, Siew GJ, Levy MH, Thwar PK, Sandahl MA, Rouse JL, Perkins LC, Sudarsan AP, Pamula V, Srinivasan V, Fair RB, Griffin PB, Eckhardt AE, Pollack MG
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 sub-microliter sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, 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 (2, 3, or 4 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.
PMID: 21932784 [PubMed - as supplied by publisher]
More...
Droplet-Based Pyrosequencing Using Digital Microfluidics.
Anal Chem. 2011 Sep 20;
Authors: Boles DJ, Benton JL, Siew GJ, Levy MH, Thwar PK, Sandahl MA, Rouse JL, Perkins LC, Sudarsan AP, Pamula V, Srinivasan V, Fair RB, Griffin PB, Eckhardt AE, Pollack MG
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 sub-microliter sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, 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 (2, 3, or 4 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.
PMID: 21932784 [PubMed - as supplied by publisher]
More...