title: Electrophoretic behavior of plasmid DNA in the presence of various intercalating dyes.
abstract: In the present study, the electrophoretic behavior of linear, supercoiled and nicked circular plasmid DNA in the presence of various intercalating dyes was characterized using pGL3 plasmid DNA as a model. The enzymatic digestion of pGL3 plasmid DNA with HindIIIwas monitored by capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). Nicked circular plasmid DNA was found to be relatively sensitive to enzymes, and was almost digested into the linear conformer after 10-min incubation, indicating that nicked circular plasmid DNA has little chance of targeting and entering the cell nucleus. Partly digested plasmid DNA containing only linear and supercoiled conformers can be used as a standard to confirm the migration order of plasmid DNA. In methylcellulose (MC) solution with YO-PRO-1 or YOYO-1, linear plasmid DNA eluted first, followed by supercoiled and nicked plasmid DNA, and nicked plasmid DNA eluted as a broad peak. With SYBR Green 1, nicked plasmid DNA eluted first as three sharp peaks, followed by linear and supercoiled plasmid DNA. The nuclear plasmid DNA from two transfected cell lines was successfully analyzed using the present procedure. Similar results were obtained with an analysis time of seconds using microchip electrophoresis with laser-induced fluorescence detection (mu-CE-LIF). To our knowledge, these results represent the first reported analysis of nuclear plasmid DNA from transfection cells by CE-LIF or mu-CE-LIF without pre-preparation, suggesting that the present procedure is a promising alternative method for evaluating transfection efficiency of DNA delivery systems.
authors.affiliation: Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Hayashi-cho 2217-14, Takamatsu 761-0395, Japan.
authors.forename: Fuquan
authors.initials: F
authors.lastname: Dang
authors.forename: Wenhao
authors.initials: W
authors.lastname: Li
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Mohammad
authors.initials: M
authors.lastname: Jabasini
authors.forename: Tatsuhiro
authors.initials: T
authors.lastname: Ishida
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kiwada
authors.forename: Noritada
authors.initials: N
authors.lastname: Kaji
authors.forename: Manabu
authors.initials: M
authors.lastname: Tokeshi
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Baba
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry; D004396:Coloring Agents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D046209:Electrophoresis, Microchip; D006801:Humans; D007364:Intercalating Agents / Q000737:chemistry*; D051379:Mice; D010957:Plasmids; D013050:Spectrometry, Fluorescence
chemical_list: D004396:Coloring Agents; D007364:Intercalating Agents; D004247:DNA