Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for high-throughput DNA sequencing.
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.
Tan
H
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Ames Laboratory-USDOE, Iowa, USA.
Yeung
E S
ES
D000818:Animals; D001331:Automation; D001483:Base Sequence; D002853:Chromatography, Liquid; D004247:DNA / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D008969:Molecular Sequence Data; D013698:Templates, Genetic
D004247:DNA