DNA analysis on electrophoretic microchips: Effect of operational variables

Abstract

Applicability of modern microfabrication technology to electrophoresis microchips initiated a rapidly moving interdisciplinary field in analytical chemistry. Electric field mediated separations in microfabricated devices (electrophoresis microchips) are significantly faster than conventional gel electrophoresis, usually completed in seconds to minutes. Electrophoretic separation of DNA molecules on microfabricated devices proved to have the potential to improve the throughput of analysis by orders of magnitude. The flexibility of electrophoresis microchips allows the use of a plethora of separation matrices and conditions. In this paper, we report on electric field mediated separation of fluorescent intercalator-labeled dsDNA fragments in polyvinylpyrrolidone matrix-filled microchannel structures. The separations were detected in real time by a confocal, single-point laser-induced fluorescence/photomultiplier setup. Effects of the sieving matrix concentration (Ferguson plot), migration characteristics (reptation plot), separation temperature (Arrhenius plot), as well as applied electric field strength and intercalator concentration on the separation of DNA fragments are thoroughly discussed.

Polymer sieving matrices in microanalytical electrophoresis

“…72,74 Because of these attractive properties, numerous studies adapt the polymer solution used in CGE to microuidic electrophoresis formats. One of the most commercially successfully microchip electrophoresis platforms (i.e., Agilent Bioanalyzer 2100) employs a PDMA-based replaceable polymer solution.…”

Highly efficient separation of dsDNA fragments on glass chips by using an ultralow viscosity sieving matrix

“…Better resolution was obtained when 0.5 lM SyTox Orange was added to the sample, which showed that the use of 0.5 lM complexing dye in the sample provided efficient labeling and separation of the DNA fragments. It was also noted that the final concentration of DNA fragments in our experiments was 5 ng/lL, the ratio of base pairs to dye was about 15:1, in contrast to the report published by Guttman's group (in which the ratio of base pairs to dye was about 1:1) [7].…”

“…The first successful efficient separation of dsDNA fragments on electrophoresis microchips was achieved in a cellulose derivative by Woolley [3]. Since then, microchip-based electrophoresis has made rapid advances in DNA analysis, such as PCR products [4 -6], double-stranded DNA fragments [3,7], and the sequencing of DNA [8,9], due to its high performance, speed, and resolution.…”

Biomedical applications of capillary electrophoresis with laser‐induced fluorescence detection

“…CE miniaturization will continue either through reducing column dimensions or placing entire electrophoresis systems on planar microfluidic chips [165,[173][174][175][176][177][178][179]. The electrophoretic separations in microchips significantly faster than the conventional separations, will have an effect especially in DNA separations [180]. The miniaturization requires smaller volume load with high sensitivity detection.…”