Pang H M, Yeung E S
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
Nucleic Acids Res. 2000 Aug 1;28(15):E73. doi: 10.1093/nar/28.15.e73.
An integrated system with a nano-reactor for cycle-sequencing reaction coupled to on-line purification and capillary gel electrophoresis has been demonstrated. Fifty nanoliters of reagent solution, which includes dye-labeled terminators, polymerase, BSA and template, was aspirated and mixed with the template inside the nano-reactor followed by cycle-sequencing reaction. The reaction products were then purified by a size-exclusion chromatographic column operated at 50 degrees C followed by room temperature on-line injection of the DNA fragments into a capillary for gel electrophoresis. Over 450 bases of DNA can be separated and identified. As little as 25 nl reagent solution can be used for the cycle-sequencing reaction with a slightly shorter read length. Significant savings on reagent cost is achieved because the remaining stock solution can be reused without contamination. The steps of cycle sequencing, on-line purification, injection, DNA separation, capillary regeneration, gel-filling and fluidic manipulation were performed with complete automation. This system can be readily multiplexed for high-throughput DNA sequencing or PCR analysis directly from templates or even biological materials.
已展示出一种集成系统，该系统带有用于循环测序反应的纳米反应器，并与在线纯化和毛细管凝胶电泳相结合。将五十纳升包含染料标记终止剂、聚合酶、牛血清白蛋白和模板的试剂溶液吸入并在纳米反应器内与模板混合，随后进行循环测序反应。然后，反应产物通过在50摄氏度下运行的尺寸排阻色谱柱进行纯化，接着在室温下将DNA片段在线注入毛细管进行凝胶电泳。超过450个碱基的DNA可被分离和鉴定。使用低至25纳升的试剂溶液即可进行循环测序反应，不过读长会稍短一些。由于剩余储备溶液可无污染地重复使用，因此在试剂成本方面实现了显著节省。循环测序、在线纯化、进样、DNA分离、毛细管再生、凝胶填充和流体操作等步骤均实现了完全自动化。该系统可轻松实现多路复用，用于直接从模板甚至生物材料进行高通量DNA测序或PCR分析。
