Capillary gel electrophoresis with laser‐induced fluorescence of plasmid DNA in untreated capillary

Abstract

A technique utilizing CGE-LIF in a bare capillary has been developed and evaluated for the detection of the three different topoisomers (linear, open circle, and supercoiled) of plasmid DNA along with the prospect of the dimer form of the supercoiled isoform. Utilizing the zwitterionic buffer, HEPES with boric acid sufficiently prevented capillary wall interactions and minimized the EOF, enabling a well-resolved separation of different plasmid isoforms. Multiple run conditions including buffer concentration and pH, hydroxypropylmethylcellulose size and amount, injection parameters, and the presence of an intercalating dye were evaluated and optimized. In addition, the feasibility of using this method as a platform for varying sizes of plasmid was investigated.

Application of Micro-Fluidic Devices for Biomarker Analysis in Human Biological Fluids

“…This technique is used when analytes that have similar charge to mass ratios need to be separated, based on just their size. (Holovics et al, 2010) The technique of choice when studying protein mixtures is capillary isoelectric focusing (CIEF). Over the past 20 years CIEF has proven to be a fast, high resolution, pI-based technique for the separation of amphoteric compounds, e.g.…”

A rapid high-resolution method for resolving DNA topoisomers

“…The separation of covalently-closed and open-circular DNA forms with capillary electrophoresis has been described previously [ 16 ], but only plasmid pUC18 was examined and the resolution of only a few topoisomers was reported. In addition, the use of capillary gel electrophoresis with laser-induced fluorescence has been described for the detection of linear, open circle, and supercoiled plasmid DNAs [ 17 ]. In this case, the resolution of relaxed DNA (from the open circle form) and the separation of individual topoisomers of different linking numbers were not reported.…”

“…In this case, the resolution of relaxed DNA (from the open circle form) and the separation of individual topoisomers of different linking numbers were not reported. In neither case was the technique suitable for use in a high-throughput format [ 16 , 17 ]. Despite these previous reports, the use of capillary gel electrophoresis has not been widely adopted for analysing topoisomers of circular DNAs and gel electrophoresis remains the predominant analytical method.…”