From http://www.nfstc.org/pdi/Subject05/pdi_s05_m01_01.htm :

... The separation medium contains a denaturant in order that the electrophoresis is conducted on single-stranded DNA fragments. Single-stranded DNA fragments are more similar to each other than double-stranded DNA fragments are to each other. Double-stranded DNA produces more electrophoretic diversity due to its three-dimensional structure.

Questions :

  1. What is the denaturant they are talking about?

  2. It's mentioned that double-stranded DNA produces more electrophoretic diversity due to its three-dimensional structure. Why should dsDNA have more 3-D structures than ssDNA? ssDNA can form hairpins and other structures. Which structures can dsDNA form?

  • 1
    $\begingroup$ As far as I see, they are talking about standard gel electrophoresis. I have done this hundrets of times for different purposes, but I have never used denaturants. I think the chance of producing unwanted 3D-structures is much bigger when it is single-stranded. This is different for RNA (which is usually not double-stranded). Here denaturating gels, as well as denaturating agent like urea or formamide in the loading buffers are used. $\endgroup$
    – Chris
    Commented Jun 8, 2014 at 6:52

2 Answers 2

  1. As already mentioned formamide can be used. However formamide has to be incubated with the nucleic acid sample and heated. Urea can be included in the gel (8%). DNA can also be denatured using NaOH. NaOH, however, cannot be used for RNA because it will cause alkaline hydrolysis.
  2. I don't much agree on this point. Though dsDNA can produce diverse structures, as buzrw mentioned, so can ssDNA (in fact more diverse). Moreover, linearization solves the issue of supercoiling. We never compare circular plasmids with each other on the gel. Long linear dsDNAs can form structures but then in such cases gel electrophoresis is not a very suitable method for comparison. PFGE might be a better option in such cases.
  1. Formamide and/or urea most likely. They both lower the melting point of the ssDNA. This allows the structures to unravel at lower temperatures. Typically you want to reach for melting temperatures around 50° C, and then run the gels at that temperature. RNA is often denatured with harsher agents, like formaldehyde because RNA form stronger structures.
  2. ds DNA can exhibit supercoiling which is a very elegant method of packaging DNA. This is a huge topic and mathematically derived. It's important is mostly due to it's role in gene expression via the interaction dsDNA has with proteins it wraps around-his tones.

Note that ds-supercoiled-DNA forms two structures; a plectoneme or a toroid, and can also have combinations of these two. It's interesting stuff. Google "DNA supercoiling" with an image search and look for a figure that shows the different levels of winding and wrapping:

I'm not very familiar with this source but the figure looks to be on the up-and-up.

Good question


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