The Hitachi SV1100 utilizes capillary electrophoresis on a microchip that is capable of rapidly sizing DNA fragments. Reproducibility of electrophoresis in different channels was shown by comparing the migration times of the internal controls, DNA fragments of 100 and 800 bp. The range of DNA sizing for this microchip is between 100 and 800 bp, and accuracy in sizing of a 322 bp DNA fragment of a pUC118 PvuII digest was observed, independent of DNA concentration. Although relatively good quantification of this fragment was observed with a DNA concentration of 1.83 ng.microL(-1), error increased in a dose-dependent manner. Furthermore, the feasibility of sequential analysis with this microchip was shown by the reproducibility of successive electrophoreses of the internal control in one channel. When the pUC118 PvuII digest was treated with endonuclease KpnI on the microchip for 10 min, sequential analysis showed that the 322 bp fragment completely disappeared and two peaks corresponding to the 130 and 192 bp fragments appeared. This analysis was performed within 4 min, and the peaks were estimated as 127 and 183 bp, respectively. These results indicate the potential of on-microchip endonuclease treatment of plasmid DNA with sequential analysis, offering high resolution in a short time.
We have developed a separation technique for DNA-protein complex based on electrophoretic mobility shift assay (EMSA) by microchip electrophoresis, which we call microchip electrophoretic mobility shift assay (μEMSA). To evaluate the μEMSA, we employed recombinant human nuclear factor-κB (rhNF-κB) and its consensus double-stranded oligonucleotide (dsOligo) fluorescently labeled with Cy5. We carried out the electrophoretic separation of the consensus dsOligo-rhNF-κB complex and the unbound dsOligo in methylcellulose solution and confirmed rapid (∼200 s) and reliable identification and semi-quantitation of the specific interaction between dsOligo and rhNF-κB. The binding specificity of rhNF-κB was confirmed by introducing non-fluorescently labeled consensus oligonucleotide as a competitor. The progression of the binding reaction under various incubation times was monitored, and it was found that the dsOligo and rhNF-κB complex formation reached equilibrium (ca. 90% of the dsOligo was bound to rhNF-κB) after 5 min. Furthermore, without any purification process, even crude NF-κB in nuclear extracts from HeLa cells was specifically detected within 120 s by the μEMSA.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.