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Detection of supercoiled and linear plasmid DNA
|Adriana Palinska , Ewelina Krawczyk , Hanna Elzanowska , Magdalena Maj-Zurawska|
Warsaw University, Department of Chemistry, Pasteura 1, Warsaw 02-093, Poland
Even with highly sensitive voltammetric or potentiometric techniques [1, 2], the detection of weak plasmid DNA signals is problematic. Moreover, plasmid DNA electrochemistry has been mainly studied using reduction signals of the nucleic acid bases on mercury electrodes , and rarely the oxidation signals on carbon electrodes . In our initial studies [3-5], we have used methylene blue (MB) entrapment into the superhelical form of pUC19 plasmid [3, 4], and/or deposition of palladium  to enhance the oxidation of the pUC19 plasmid. The plasmid layer was adsorbed on glassy carbon electrode, achieving an almost ten fold increase of the DNA signals.
In this work, we have focused on the detection of the oxidation signals of two types of plasmid DNA – pUC19 and pGEX-4T-2 in two forms – superhelical and linear. The oxidation of these two plasmids in two different forms can be distinguished by their slow adsorption on glassy carbon (GC) electrode from 0.25 M acetic buffer pH 4.7 containing 18 mM MgCl2 and low concentrations (pg/mL) of the plasmids. The adsorption of the plasmids was monitored using SW, DP and AC voltammetry. Smaller (2686 bp) supercoiled pUC19 accumulated faster than larger (4970 bp) supercoiled pGEX-4T-2. Even though both forms of plasmid consisted of the same number of base pairs, we have noticed significant differences between the accumulation of supercoiled and linear forms of these plasmids. Only one broad signal of the oxidation of nucleic acid bases was detected for the linear plasmid, instead of typically observed two signals corresponding to guanine and adenine oxidation.
In the presence of methylene blue, a typical intercalator , the oxidation signals of bases are significantly enhanced [3-5], allowing for the detection of the plasmids at concentrations lower than pg/mL. Except for a typical MB signal at -0.2 V vs. SCE, MB(I), another MB oxidation signal at ca. 0.95 V, MB(II), is used to distinguish not only between two different plasmids, but also between their supercoiled and linear forms. The MB(I) signal, typically used to probe DNA intercalation , is low or even hardly visible at MB concentrations below 1-2 µM, while in the same MB concentration range, the MB(II) signal increases significantly. The AC voltammetric experiments sensitive to plasmid DNA adsorption in a wide potential range from -0.6 v to 1.4 V suggest that the interactions of MB are related to the capacity and resistance changes of the plasmid layer.
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Presentation: Poster at SMCBS'2011 International Workshop, by Adriana Palinska
See On-line Journal of SMCBS'2011 International Workshop
Submitted: 2011-09-08 01:19 Revised: 2011-09-08 01:19