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Nucleic Acids Research, 2000, Vol. 28, No. 15 E73-e73
© 2000 Oxford University Press

Automated one-step DNA sequencing based on nanoliter reaction volumes and capillary electrophoresis

Ho-ming Pang and Edward S. Yeung*

Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, IA 50011, USA

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°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.

* To whom correspondence should be addressed at: Department of Chemistry, Iowa State University, Ames, IA 50011, USA. Tel: +1 515 294 8062; Fax: +1 515 294 0266; Email: yeung@ameslab.gov


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