Conformation-sensitive gel electrophoresis

Very valuable information on the energy and conformation characteristics of ccDNA has arisen from experiments in which the value of Lk could vary, and the equilibrium distribution of the cc molecules over the Lk value was studied. The most convenient way to vary Lk is to employ special enzymes we have already mentioned above, the topoisomerases. The studies under discussion employed type I topoisomerases, which alter the topological state of ccDNA by breaking and rejoining only one of the strands of the double helix. The mechanism of action of these enzymes has been elaborated in great details (Wang, 1996 [86]). These enzymes relax the distribution of the molecules over the Lk value to its equilibrium state. The very sensitive gel- electrophoresis method was used to analyze the distribution of the ccDNA molecules over the Lk value. This method can easily separate two molecules of ccDNA that differ in Lk just by one. 

Figure 6-5. Rapid and sensitive methods for detecting mutations in DNA. (a) Denaturing gradient gel electrophoresis. (b) Single-strand conformation polymorphism.

Several rapid and sensitive methods have been developed to detect mutations in cDNA and genomic DNA. These include ribonuclease (RNase) protection analysis, denaturing gradient gel electrophoresis, and single-strand conformation polymorphism analysis. These methods can be used in conjunction with the PCR technique to detect small deletions or insertions and single base substitutions. 

In the Eastern-Western procedure, lipids are first applied to a solid support such as nitrocellulose. Next, proteins subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis are transferred by standard Western blotting techniques to the solid support in such a manner that the protein of interest is transferred to the lipid patch. During electrotransfer of protein to the solid support, protein, lipid, and sodium dodecyl sulfate mix and as transfer continues the sodium dodecyl sulfate is removed leaving behind the protein to refold in the presence of lipid. Attachment of the refolded protein to a solid support allows one to probe protein structure using conformation-sensitive antibodies or protein function... 

Ionic interactions are also used to associate genetic material with nanoparticulate carriers. Electrophoresis is perhaps the easiest way to assess the association efficiency of nucleic acids to a nanostructure. The associated genetic material will not migrate in an electrophoresis gel as would happen with free nucleic acids. It is also useful to visually determine the conformation of the plasmid DNA, supercoiled being the most effective form in comparison to circular and open forms. The association efficiency can be quantitatively determined using commercial kits and fluorescence techniques or, directly but with a lower sensitivity, by UV determination. For this purpose, the free nucleic acid needs to have been previously separated and subsequently quantified. 

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