Sensitive Analysis of Topoisomers of Large Plasmid DNA by Capillary Gel Electrophoresis With Laser‐Induced Fluorescence in Uncoated Capillary

This study presents a capillary gel electrophoresis-laser-induced fluorescence (CGE-LIF) method for analyzing topoisomers of large plasmid DNA (up to 19 kbp) using an uncoated capillary. Separation of large plasmid isoforms, particularly the open circular (OC) form, poses significant challenges in gel buffer formulation. To address this, we systematically investigated the effects of multiple parameters-including polymer type, fluorescent dye selection, stir bar geometry, stirring speed and duration, gel volume, container dimensions, and technique for polymer addition-on separation performance. The optimized gel buffer composition was 0.9% polyethylene oxide (PEO), 0.02% SYBR Green, 50 mM N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), 1 mM EDTA, and 20 mM NaCl at pH 6.25. Replacing hydroxypropyl methyl cellulose (HPMC) gel with PEO gel significantly improved OC isoform separation. Furthermore, substituting SYBR Gold with SYBR Green enhanced the robustness of OC separation and yielded a 4- to 8-fold increase in sensitivity. The PEO gel preparation process was successfully scaled and optimized at the 100 mL level. With consistent capillary cleaning and storage protocols, the method demonstrated excellent reproducibility over 150 consecutive runs.