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Electrophoretic mobility of supercoiled, catenated and knotted DNA molecules

2014; Oxford University Press; Volume: 43; Issue: 4 Linguagem: Inglês

10.1093/nar/gku1255

1362-4962

Jorge Cebrián, Maridian J. Kadomatsu-Hermosa, Alicia Castán, Víctor Martínez, Cristina Parra, María José Fernández-Nestosa, Christian E. Schaerer, María-Luisa Martínez-Robles, Pablo Hernández, Dora B. Krimer, Andrzej Stasiak, Jorge B. Schvartzman,

Nanopore and Nanochannel Transport Studies

We systematically varied conditions of twodimensional (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 electrophoresis.