Capillary gel electrophoresis with laser-induced fluorescence of plasmid DNA in untreated capillary.
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.
Holovics
Heidi J
HJ
Analytical R&D, Pfizer BioTherapeutics Pharmaceutical Sciences R&D, Chesterfield, MO, USA. heidi.holovics@pfizer.com
He
Yan
Y
Lacher
Nathan A
NA
Ruesch
Margaret N
MN
D001888:Boric Acids; D004247:DNA / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D006531:HEPES; D007834:Lasers; D010957:Plasmids / Q000302:isolation & purification*
D001888:Boric Acids; D004247:DNA; C032688:boric acid; D006531:HEPES