Droplet-based pyrosequencing using digital microfluidics.
Droplet-based pyrosequencing using digital microfluidics.
复制标题
DOI：
10.1021/ac201416j
复制
发表时间：
2011-11-15
影响因子：
7.4
通讯作者：
Pollack, Michael G.
中科院分区：
文献类型：
--
作者：
Boles, Deborah J.;Benton, Jonathan L.;Siew, Germaine J.;Levy, Miriam H.;Thwar, Prasanna K.;Sandahl, Melissa A.;Rouse, Jeremy L.;Perkins, Lisa C.;Sudarsan, Arjun P.;Jalili, Roxana;Pamula, Vamsee K.;Srinivasarn, Vijay;Fair, Richard B.;Griffin, Peter B.;Eckhardt, Allen E.;Pollack, Michael G.
报告了使用基于电润湿的数字微流体在液滴内实施焦磷酸测序化学的可行性。在印刷电路板上图案化的电极阵列用于控制充油室内包含的亚微升大小的液滴的形成、运输、合并、混合和分裂。实施三酶焦磷酸测序方案，其中单个液滴含有酶、脱氧核糖核苷酸三磷酸（dNTP）和DNA模板。将DNA模板锚定到磁珠上，这使得它们能够在核苷酸添加之间被彻底洗涤。对试剂和方案进行优化，以使背景信号、响应线性、循环效率和洗涤效率最大化。作为可行性的初步证明，使用从头方案和重测序方案对229 bp近平滑念珠菌模板的一部分进行测序。基于原始热解图水平，重测序方案产生了超过60 bp的序列，具有100%的序列准确度。对于C中包含的所有均聚物（两个、三个或四个核苷酸）观察到极好的线性。近平滑序列随着微流体设计的改进，预期更长的读取、更高的通量和改进的过程集成（即，“样品到序列”能力）最终可以使用这种低成本平台来实现。
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.
登录
查看更多内容
影响因子：
9.4
作者：
Borman, Andrew M.;Linton, Christopher J.;Johnson, Elizabeth M.
通讯作者：
Johnson, Elizabeth M.
影响因子：
6.1
作者：
Moon, Hyejin;Wheeler, Aaron R.;Kim, Chang-Jin CJ''
通讯作者：
Kim, Chang-Jin CJ''
影响因子：
2.7
作者：
Cho, SK;Moon, HJ;Kim, CJ
通讯作者：
Kim, CJ
影响因子：
6.1
作者：
Sista RS;Eckhardt AE;Srinivasan V;Pollack MG;Palanki S;Pamula VK
通讯作者：
Pamula VK
影响因子：
4
作者：
Pollack, MG;Fair, RB;Shenderov, AD
通讯作者：
Shenderov, AD