Tan H, Yeung E S
Ames Laboratory-USDOE, Iowa, USA.
Anal Chem. 1998 Oct 1;70(19):4044-53. doi: 10.1021/ac980406i.
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 approximately 60 degrees C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at approximately 70 degrees 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.
基于多重微流控和毛细管阵列电泳，已展示了一种从DNA模板到其一级序列的集成式多重在线仪器。该仪器以并行方式自动处理八个模板，依次进行反应、纯化、变性、预浓缩、进样、分离和检测。利用多重冻融切换原理和分配网络来管理流动和样品传输。在热空气热循环仪的八毛细管阵列中进行染料标记的终止子循环测序反应。随后，测序梯直接加载到在约60摄氏度下运行的单独尺寸排阻色谱柱中进行纯化。在约70摄氏度的交叉组件处同时完成用于毛细管电泳的在线变性和堆积进样。不仅分离毛细管阵列，而且反应毛细管阵列和纯化柱在每次运行后都可以再生。原始数据允许在460 bp的长度内进行碱基识别，准确率达98%。该系统可扩展至96毛细管阵列，不仅将有利于高速、高通量DNA测序，还将有利于基因分型。
