Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for high-throughput DNA sequencing.
Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for high-throughput DNA sequencing.
复制标题
多重在线样品制备与毛细管电泳的自动化和集成，用于高通量 DNA 测序。
DOI：
10.1021/ac980406i
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发表时间：
1998
影响因子：
7.4
通讯作者：
Yeung,ES
中科院分区：
文献类型：
--
作者：
Tan,H;Yeung,ES
基于多重微流体和毛细管阵列电泳，已经证明了一种从 DNA 模板开始到其一级序列的集成多重在线仪器。仪器自动并行处理八种模板，包括反应、纯化、变性、预浓缩、进样、分离和检测。利用多重冷冻/解冻切换原理和分配网络来管理流量和样品运输。染料标记的终止子循环测序反应在热风热循环仪的八毛细管阵列中进行。随后，将测序梯直接加载到在〜60°C下操作的单独的尺寸排阻色谱柱中进行纯化。毛细管电泳的在线变性和堆积注射在〜70°C的交叉组装装置上同时完成。不仅分离毛细管阵列，反应毛细管阵列和纯化柱也可以在每次运行后再生。原始数据允许碱基检出高达 460 bp，准确度为 98%。该系统可扩展至 96 个毛细管阵列，不仅有利于高速、高通量 DNA 测序，还有利于基因分型。
An integrated and multiplexed on-line instrument starting from DNA templates to their primary sequences has been demonstrated based on multiplexed microfluidics and capillary array electrophoresis. The instrument automatically processes eight templates through reaction, purification, denaturation, preconcentration, injection, separation, and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were utilized to manage flow and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an eight-capillary array in a hot-air thermal cycler. Subsequently, the sequencing ladders are directly loaded into separate size exclusion chromatographic columns operated at ∼60 °C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at ∼70 °C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. The raw data allow base calling up to 460 bp with an accuracy of 98%. The system is scalable to a 96-capillary array and will benefit not only high-speed, high-throughput DNA sequencing but also genetic typing.