Automated one-step DNA sequencing based on nanoliter reaction volumes and capillary electrophoresis.
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.
Pang
H M
HM
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
Yeung
E S
ES
D001331:Automation; D002850:Chromatography, Gel / Q000379:methods; D003365:Costs and Cost Analysis; D004247:DNA / Q000302:isolation & purification; D019075:Electrophoresis, Capillary; D017422:Sequence Analysis, DNA / Q000191:economics / Q000295:instrumentation / Q000379:methods*
D004247:DNA