Analysis of laser-induced plasmic DNA photolysis by capillary electrophoresis.
Capillary electrophoresis (CE) was used to monitor the laser-induced conversion of supercoiled pKOL8UV5 plasmid DNA into nicked conformers. The plasmid samples (0.1 mg/ml) were incubated in the absence or presence of 110 mumol/l ethidium bromide (EB) and then exposed to 100 J of argon laser radiation (488 nm). The nicked, open circular conformers were separated from the supercoiled DNA by a 15% increase in retention time. Approximately 90% of the control DNA was in the supercoiled form. Laser radiation in the presence of EB caused complete conversion of the supercoiled plasmid DNA into nicked conformers. Laser-induced fluorescence CE (LIF-CE) was about 100-fold more sensitive than UV-CE in the detection of these conformers. Agarose gel electrophoresis confirmed these findings and showed the presence of the nicked plasmid conformers. Based on these comparisons, CE is an efficient analytical tool for the identification of laser-induced conformational changes in plasmid DNA.
Nackerdien
Z
Z
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Morris
S
S
Choquette
S
S
Ramos
B
B
Atha
D
D
D004249:DNA Damage; D004274:DNA, Recombinant / Q000378:metabolism / Q000528:radiation effects*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007834:Lasers; D010782:Photolysis; D013056:Spectrophotometry, Ultraviolet
D004274:DNA, Recombinant