Interpretative diagrams are shown to the right. In these diagrams and the following figures supercoiled dimers CCCd are called ScDimers and are depicted in dark blue, nicked-casemated monomers CatAm are called CatAs and are depicted in red, and nicked-knotted dimers Knd are called KnDimers and are depicted in pink. Electrophoresis conditions are detailed to the left and interpretative diagrams are shown to the right.
OCd refers to the mobility of dimeric open circles used to align the different immunograms. Comparison of the electrophoretic mobility of different topoisomers during the first and second dimension of 2D gels where the latter occurred at different voltages. The actual numbers are shown in Supplementary Tables S1—S4. Comparison of the electrophoretic mobility of different topoisomers during the first and second dimension of 2D gels where the latter occurred in gels of different agarose concentrations.
Comparison of the relative electrophoretic mobility of different topoisomers during the first and second dimension of 2D gels where the latter occurred at different voltages. Comparison of the relative electrophoretic mobility of different topoisomers during the second dimension of 2D gels that occurred in gels of different agarose concentrations. The actual numbers are shown in Supplementary Tables S5 and S6. Their relevant genotypes are detailed below:. Competent cells were transformed with monomeric bp or dimeric forms bp of pBR18, a derivative of pBR where the tetracycline resistance promoter was replaced with the poly-linker of pUC18 Isolation of plasmid DNA was performed as described elsewhere 25 , In all cases the DNA samples analyzed were prepared from separately isolated pools of supercoiled DNAs mixed in vitro with nicked forms of knotted or catenated molecules.
To induce single-stranded breaks, DNA was digested with Nb. The first dimension was in a 0. The second dimension was in a 0. The dissolved agarose was poured around the excised agarose lane from the first dimension and electrophoresis was at 5—6. Southern transfer was performed as described before Labeled DNA was added and hybridization lasted for 12—16 h. To obtain broad spectrum of topoisomers of supercoiled dimeric forms of pBR18 we transformed E.
A similar result could be obtained using chloroquine 2D gels. However, chloroquine would not have helped to separate nicked DNA forming different knots and catenanes since in these molecules the DNA is not under a torsional constraint. The aim of our investigation was to have a good way of separating various topological forms including different types of nicked knots and catenanes.
To obtain DNA preparations enriched in catenanes we transformed E. Therefore, in these cells Norfloxacin inhibits only Topo IV leading to the accumulation of catenated duplexes 34 , Once isolated this DNA was digested with the nicking enzyme Nb.
To obtain knotted dimers, we transformed E. Their electrophoretic mobility during the first and second dimensions varied slightly between experiments although the differences were not significant. The actual figures are shown in Supplementary Tables S1—S6. The electrophoretic mobility for all the families analyzed during the first dimension of the 2D gel system used, run in 0. However, this was not the case anymore for the electrophoretic runs used during the second dimension. Note that the simplest KnDimer, the trefoil knot, has three nodes 18 , On the other hand, during this second dimension, the behavior of CatAs was also curious.
This behavior expanded with increasing voltage. Precisely the opposite was observed for KnDimers. The effect of agarose concentration at a constant voltage 5. For trefoil KnDimers, their electrophoretic mobility decreased from 7. In short, the electrophoretic mobility of all topoisomers studied decreased as agarose concentration increased.
As previously mentioned, it was repeatedly shown that the electrophoretic mobility of different DNA knots and catenanes is proportional to the compactness of their unperturbed equilibrium shapes when analyzed in low concentration agarose gels run at low voltage 16 — The behavior of these topoisomers in high concentration agarose gels run at relatively high voltage received considerable less attention 3.
Bell and Byers 4 analyzed X-shaped and linear molecules in 2D gels where the first dimension run in a 0. An inversion in electrophoresis mobility was observed using a similar 2D gel version for knotted replication intermediates, linear fragments containing an internal knotted bubble 25 , 38 — Weber et al. Earlier studies demonstrated that relatively large supercoiled DNA molecules have branched structure 45 and that this may play a significant role in their electrophoretic mobility.
Here, we report that the electrophoretic mobility of CatAs, ScDimers and KnDimers with the same molecular mass systematically increased with their progressing compaction during the first dimension of a 2D gel system run in a 0. However, during the second dimension, run in a 0. We propose that when gel pore sizes become smaller than the dimensions of undisturbed equilibrium shapes of analyzed DNA molecules, the deformability and thus the easiness of the molecules to pass through the gel pores becomes the limiting factor of their electrophoretic mobility 46 — In the case of knotted or catenated DNA molecules, there are two partially opposing effects of increasing topological complexity: i A decrease of the spatial extent of the molecules measured by such observables as radius of gyration or the average inverse distance.
This is because there is more nanograms of DNA in 3 than in 2 the number of molecules in 3 must be much higher than in 2. Plasmid DNA can exist in three conformations: supercoiled, open-circular oc , and linear supercoiled plasmid DNA is often referred to as covalently closed circular DNA, ccc.
In vivo, plasmid DNA is a tightly supercoiled circle to enable it to fit inside the cell. In the laboratory, following a careful plasmid prep, most of the DNA will remain supercoiled, but a certain amount will sustain single-strand nicks. Given the presence of a break in only one of the strands, the DNA will remain circular, but the break will permit rotation around the phosphodiester backbone and the supercoils will be released. A small, compact supercoiled knot of ccc-DNA sustains less friction against the agarose matrix than does a large, floppy open circle of oc-DNA.
Linear DNA runs through a gel end first and thus sustains less friction than open-circular DNA, but more than supercoiled. Thus, an uncut plasmid produces two bands on a gel, representing the oc and ccc conformations.
If the plasmid is cut once with a restriction enzyme, however, the supercoiled and open-circular conformations are all reduced to a linear conformation. Following isolation, spontaneous nicks accumulate as a plasmid prep ages. This can clearly be seen on gels as the proportion of the two conformations change over time: plasmids preps that have been thawed and refrozen many times, show more oc DNA than fresh preps.
This is a black-and-white photograph of an agarose gel containing ethidium bromide, after electrophoresis of three DNA samples. It looks like you're using Internet Explorer 11 or older. This website works best with modern browsers such as the latest versions of Chrome, Firefox, Safari, and Edge. If you continue with this browser, you may see unexpected results. Introduction There are a number of reasons why a restriction enzyme may not cut efficiently. Supercoiled plasmids In order to visualize the results of the experiment, we will perform agarose electrophoresis on our digested samples.
Supercoiled plasmid bands on a gel In gel electrophoresis of DNA, we normally consider the migration speed of a piece of DNA to depend primarily on its size unlike proteins which have a migration speed that can also be significantly affected by the pH of the gel. Subjects: Biological Sciences. You may republish or adapt this guide for educational purposes, as long as proper credit is given.
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However, you see more than one band on your digested sample and you wonder which one to cut. In this article, we review the different forms of plasmid DNA and offer some useful tips to interpret your gel. Agarose, produced from seaweed, is a polysaccharide agar. During polymerization, agarose polymers link non-covalently and form a network of bundles. This network consists of pores with molecular filtering properties. DNA separation occurs due to the mesh-like nature of the agarose gel.
Smaller DNA fragments can move quickly through the pores, while larger fragments get caught and therefore travel slowly. Under a powerful microscope, a gel will look porous, but to the naked eye, it looks like a smooth, opaque gelatin in the shape of a square with wells near one end of the surface.
A well is a hollow pocket in the gel where the DNA is loaded. Because of the negatively charged phosphate backbone, DNA holds a slight negative charge that allows the molecule to migrate to the positively charged anode. The travel distance of DNA molecules within an agarose gel is proportional to the log of its molecular weight.
The gel electrophoresis conditions including the presence of ethidium bromide, gel concentrations, electric field strength, temperature, and ionic strength of the electrophoresis buffer may affect the mobility of the plasmid DNA. Due to the net-like nature of agarose gel, circular plasmid DNA is caught up easier in the agarose mesh.
The electrophoretic trapping is a balance between the electrophoretic force pulling the circular plasmid DNA against the trap and diffusion allowing the circular plasmid DNA to escape a trap. So, large circular molecules have a greater chance to get trapped than smaller DNA.
CCC monomer is a negatively charged and supercoiled plasmid. Intact supercoiled plasmids have compact double-stranded DNA twisted around itself. Undigested plasmid DNA are usually supercoiled. An open circular form is caused by the nicking cleavage of one DNA strand.
UV irradiation or nucleases can cause this single-strand break. This structure is a relaxed and less compact form of plasmid. It also has less supercoiling than the CCC form.
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