Swerdlow H, Jones B J, Wittwer C T
Department of Human Genetics, University of Utah, Salt Lake City 84112, USA.
Anal Chem. 1997 Mar 1;69(5):848-55. doi: 10.1021/ac961104o.
A simple, reliable, automated genetic analysis instrument has been designed and prototyped. The system uses novel fluidic technology, coupling thermal cycling, reaction purification, in-line loading, and capillary electrophoresis in a single instrument. Samples in the loop of an injection valve are amplified inside a rapid air thermal cycler. A liquid chromatographic separation eliminates contaminants and excess salt. The sample is loaded in an efficient, continuous, flow-through manner onto a polymer-filled separation capillary. Detection by laser-induced fluorescence produces signal-to-noise ratios of 1000:1 or greater. Refilling of the polymer-filled capillary is automatic; during the run, the system is reconditioned for injection of another sample. Since all components and connections are fluidic, automation is natural and simple. The instrument is reliable and fast, performing PCR reaction cycling, purification and analysis, all in 20 min. Reproducibility (CV) of retention times is 2% (n = 129) and of peak areas 9% (n = 34). Bubbles and particulates are eliminated by the chromatography column. Adaptation of the instrument prototype for separation of DNA-sequencing reactions is described; cycle sequencing and electrophoresis of a single lane are complete in 90 min. Implications and challenges for development of fully automated fluidic instruments for genomic sequencing are discussed.
一种简单、可靠的自动化基因分析仪器已设计完成并制作出了原型。该系统采用了新型流体技术，在一台仪器中集成了热循环、反应纯化、在线进样和毛细管电泳功能。进样阀定量环中的样品在快速空气热循环仪内进行扩增。液相色谱分离可去除污染物和过量盐分。样品以高效、连续、流通的方式加载到填充聚合物的分离毛细管上。激光诱导荧光检测产生的信噪比为1000:1或更高。填充聚合物的毛细管自动重新填充；在运行过程中，系统会进行重新调节以注入另一个样品。由于所有组件和连接都是流体的，自动化自然且简单。该仪器可靠且快速，能在20分钟内完成PCR反应循环、纯化和分析。保留时间的重现性（CV）为2%（n = 129），峰面积的重现性为9%（n = 34）。色谱柱可消除气泡和颗粒。描述了该仪器原型用于DNA测序反应分离的适应性；单泳道的循环测序和电泳在90分钟内完成。讨论了开发用于基因组测序的全自动流体仪器的意义和挑战。
