Evaluation of DNA Fragment Sizing and Quantification by the Agilent 2100 Bioanalyzer

Abstract

Capillary electrophoresis (CE) achieves efficient separation of molecular species by the application of high voltages to samples in solution (1). Commercial CE units, available for slightly more than a decade, have found numerous applications (2)(3)(4)(5)(6), but are expensive (∼$60 000) and require substantial user training and experience. Recent advances have allowed CE to be performed on microchip devices (7)(8)(9)(10)(11). We evaluated the Agilent 2100 Bioanalyzer (Agilent Technologies), which represents a new generation of CE instruments that use this technology. The Bioanalyzer is relatively inexpensive (∼$18 000) and is simple to operate, requiring only routine pipetting and basic computer skills. Typically, 12 nucleic acid samples can be sized and quantified on a disposable chip within 30 min. Chips are fabricated from glass and comprise an interconnected network of fluid reservoirs and microchannels, which must be filled with a gel-dye mixture. Each chip contains 16 wells: 3 for loading the gel-dye mixture, 1 for a molecular size ladder, and 12 for experimental samples. The movement of nucleic acids through the microchannels is controlled by a series of electrodes, each of which is independently connected to a common power supply. The Bioanalyzer displays data as both migration-time plots and as computer-generated virtual gels. Traditional CE operating variables [temperature, voltage, capillary material, and pH, ionic strength, and viscosity of buffer (12)] cannot be modified. The instruments costs ∼$18 000, and chips cost ∼$12–18 per chip ($1–1.50 per sample). The gel-dye mixture consists of a linear polymer and a fluorescent, intercalating dye. The marker mixture consists of a buffer along with lower and upper molecular size markers, which the Bioanalyzer uses as references when sizing DNA fragments. The upper marker is also used as a reference for calculating the …