Molecular Biology


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0 Q&A 8925 Views Aug 5, 2016
Capillary electrophoresis (CE) is identified as a promising technology for the study of nucleic acid molecules because of its high efficiency, high throughput with automation and integration. Compared to the traditional method of slab gel electrophoresis (SGE), the advantages of CE cannot be emphasized more. Most of CE process, including sample injection, detection and data analysis, is able to be automated which will save great labor for industrial and research labs. CE used the separation channel with micrometer-scale diameter, so the joule heat is easy to be dissipated during electrophoresis. Thus high separation voltage (> 100 V/cm) is allowed in CE while in SGE (usually ~10 V/cm) it usually causes severe band broadening. Because the band broadening is restrained efficiently in CE, it is capable of detecting minute samples and becoming more sensitive than SGE. The advantage of allowing high voltage consequently speeds up the CE separation and yields a better throughput compared to SGE. CE costs less reagents, for example buffer solutions, sieving matrix, dye reagents etc. In addition, the micrometer-scale channel is easy to be integrated with upstream and downstream sample treatment units, forming a lab on a chip. This merit of CE already attracted considerable interests among researchers from various areas.

The difficulties of CE involve filling the gels (agarose or cross-linked polyacrylamide) into the capillary tube. Also, the reproducibility and the life-time of the gel-capillary are limited. But the small-diameter capillary allows to use replaceable polymer solutions, which can efficiently prevent the convection of the separation buffer. Polymer solutions are easier to be filled into the capillary and yield more stable separations. Thus, those difficulties are resolved by doing capillary polymer electrophoresis (CPE), which is going to be described in this protocol.

Several separation modes, for example, capillary gel electrophoresis (CGE), CPE, capillary zone electrophoresis (CZE), capillary isotachophoresis (CITP) and so on, have been developed for analysis of different kinds of molecules. Here, we introduce the protocol for CPE in detail, which is for the separation of dsDNA, dsRNA (including siRNA) molecules. Polymer solutions are filled into the capillary as a sieving matrix for double strand nucleic acids separation. Hydroxyethylcellulose (HEC) polymer is employed as the sieving polymer in this case. A home-built CE system is described in detail.

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