Evaluation of DNA Fragment Sizing and Quantification by the Agilent 2100 Bioanalyzer
2000; American Association for Clinical Chemistry; Volume: 46; Issue: 11 Linguagem: Inglês
10.1093/clinchem/46.11.1851
ISSN1530-8561
AutoresNicholas J. Panaro, Po Ki Yuen, Taku Sakazume, Paolo Fortina, Larry J. Kricka, Peter Wilding,
Tópico(s)Microfluidic and Bio-sensing Technologies
ResumoCapillary 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 …
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