Few fluorescent dyes are reported to differentiate between supercoiled plasmid DNA and relaxed DNA by differential binding of the dye upon DNA relaxation ( ex: Picogreen). I was wondering how to have differential binding of intercalating dye upon DNA supercoiling/relaxation as this process would not change the nature of DNA grooves that it binds to ?
Differentiating between supercoiled and relaxed plasmid by fluorescent dye intercalation is not a particularly sensitive method, for the reason you stated probably. Other methods have been described based on introducing special sequences into the plasmids. Here are some examples:
Article A Homogeneous, High-Throughput Fluorescence Anisotropy-Based...
Article High-throughput assays for DNA gyrase and other topoisomerases
Article Fluorescently labeled circular DNA molecules for DNA topolog...
Thanks Adam for your feedback. I think you are correct about the sensitivity issue. However, I am not looking for which method would be more sensitive. I am more concerned about key structural/conformational differences between supercoiled vs relaxed plasmid that would allow intercalative dye to differentiate between them.
Perhaps you will find what you need in this article:
http://ase.tufts.edu/biology/labs/mirkin/documents/Mirkin_DNA-topology.pdf
This article is about using ethidium bromide to detect supercoiling.
Article Fluorometric methods employing low concentrations of ethidiu...
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA