Droplet-Based Pyrosequencing Using Digital Microfluidics

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• 作者：Deborah J. Boles ; Jonathan L. Benton ; Germaine J. Siew ; Miriam H. Levy ; Prasanna K. Thwar ; Melissa A. Sandahl ; Jeremy L. Rouse ; Lisa C. Perkins ; Arjun P. Sudarsan ; Roxana Jalili ; Vamsee K. Pamula ; Vijay Srinivasan ; Richard B. Fair ; Peter B. Griffin ; Allen E. Eckhardt ; Michael G. Pollack
• 刊名：Analytical Chemistry
• 出版年：2011
• 出版时间：November 15, 2011
• 年：2011
• 卷：83
• 期：22
• 页码：8439-8447
• 全文大小：957K
• 年卷期：v.83,no.22(November 15, 2011)
• ISSN：1520-6882

文摘

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 <i>Candida parapsilosisi> 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 <i>C. parapsilosisi> sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., 鈥渟ample-to-sequence鈥?capability) could eventually be achieved using this low-cost platform.