Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules

We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ∼15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresi

Numerical simulation of two-dimensional agarose gel electrophoresis of knotted and supercoiled DNA molecules

"The present work introduces a mathematical model, based on conservation laws and Maxwell-Stefan equations of mass transport, to analyze the electrophoretic mobility of two topological DNA families: supercoiled and knotted DNA molecules with the same total molecular mass. Comparison of experimental results and numerical simulations showed that our model is able to accurately reproduce two-dimensional (2D) agarose gel electrophoresis of supercoiled dimers and nicked knotted dimers under different conditions of electrostatic potential. The implementation of this mathematical approach could be useful to optimize conditions for the separation of different types of DNA topoisomers during electrophoresis."CONACYT - Consejo Nacional de Ciencias y TecnologíaPROCIENCI