Non-destructive separation of the strands of
a circular duplex chromosome

If DNA really had the Watson-Crick helical twist, then it would be impossible to separate the individual single strands of a duplex circular chromosome unless at least one of them was broken open. Yet it is known that they can be separated. This was accomplished by Tai Te Wu in 1996). His publication was, in my opinion, the second-most-important DNA paper of the 20th century, exceeded in importance only by the original 1953 Watson-Crick Nature paper which first presented the double-helix.

As noted previously, the Wu experiment was very difficult, expensive and time-consuming. No one will ever attempt to repeat it. In sharp contrast, the protocol presented here is easy, inexpensive, and takes only a few minutes to complete. The only serious work involved is the identification of the product by electron microscopy or other means, which will require a few hours of work by your lab technicians.

This experiment has never been done, and therefore the outcome cannot be 100% guaranteed. Nevertheless, the logic behind it is nothing short of compelling; so much so that it is almost certain that it will succeed, as you will see when you begin to read and understand the subject of non-helical DNA structure.

Strand separation protocol

This experiment is ridiculously simple to do — other than the time in dialysis, the seven steps below, which are all that is necessary to prepare the DNA for EM study, can be completed by a skilled lab worker in a matter of minutes.

Understanding the logic behind the experiment, however, is a real challenge. The complete and convoluted history, leading to the inescapable conclusion that the strands of a circular plasmid can indeed be separated as described below, is contained in my publication Methods for Non-Destructively Separating or Reannealing the Strands of Circular Duplex DNA Chromosomes, which can be seen and read on this web site, or downloaded immediately by simply clicking the above link.

You do not have to read the manuscript, or understand the experiment, to perform it. But I can't imagine that you won't have the curiosity to want to understand what you're doing.

The protocol begins with 4 simple stock solutions:

      10 mM Tris•HCl (pH 8), 0.5 mM EDTA
       1 M NaCl
       1.1 M NaOH
       1.1 M Tris•HCl

The recommended DNA for this experiment is pBR322. At the time this page was written, this was available from Life Technologies, or from New England Biolabs. The logic behind using this DNA is that it is essential for the "sister" reannealing experiment, which is presented elsewhere on this web site. The "sister" experiment requires use of "nicking" enzymes (i.e., site-specific, strand specific nucleases) that have cleavage sites in pBR322, wherefore it is desirable that pBR322 be used for both experiments.

But it is not absolutely necessary to use pBR322. Any duplex circular plasmid chromosome should work.

The seven simple steps are as follows:

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(1) Dilute the pBR322 DNA by addition of 9 volumes of 10 mM Tris•HCl, pH 8, 0.5 mM EDTA.

Comment: The pBR322 DNA is sold at a high concentration (500 mg/ml). This step will dilute it to 50 mg/ml; a concentration in the range employed in the published alkali denaturation studies. It is wise to avoid excessively-high DNA concentrations in this work, because high concentrations of DNA predispose to unintentional reannealing and poorly-understood aggregation effects.
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(2) Add one-half volume of 1 M NaCl. (Note: This gives a new DNA concentration = [2/3] x 50 = 33.3 mg/ml).

Comment: This gives a final salt concentration of 0.33 M. This relatively high salt concentration is the salt concentration used in the reported alkali denaturation studies. Although the primary denaturant in those studies was NaOH, a high starting concentration of NaCl was employed, and it may be that the high NaCl concentration was also necessary to facilitate the irreversible alkali denaturation of the DNA described in the next step.
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(3) Add one-tenth volume of 1.1 M NaOH. (Make note of that volume for the step below). (New DNA concentration = [10/11] x 33.3 = 30.3 mg/ml).

Comment: This amount of base is precisely-calculated to raise the pH to at least 13. At that pH, your circular DNA will be irreversibly denatured to a form known as "Form IV".

You can create Form IV without knowing what it is, but that is not, in my opinion, the best way to do this experiment. It is better to know what you're dealing with. Form IV is the subject of a lengthy PowerPoint presentation on this web site, entitled Form IV – the Final Puzzle Piece. Although this is a very long presentation, it is commensurately informative, and I cannot recommend it highly enough to anyone who is truly interested in DNA structure.
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(4) Add 1.1 M Tris•HCl, an amount exactly equal in volume to the volume of NaOH added in the step above. (New DNA concentration = [11/12] x 30.3 = 27.8 mg/ml).

Comment: This will drop the pH to about 8. The exact final pH is not of critical importance. This is only done to reduce the possibility of hydrolysis of the DNA sugar-phosphate backbone by prolonged exposure to high pH.

WARNING: From this point forth, the DNA should be kept on ice. Your DNA is denatured, and you don't want it to renature under these conditions. Although the DNA concentration at this point; about 28 mg/ml; is probably not high enough to readily facilitate either unintended renaturation or aggregation, the possibility of these things must always be kept in mind. Renaturation/aggregation will be less likely if the DNA is kept cold.
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(5) Dialyze the DNA, in the cold, against the first buffer (10 mM Tris•HCl, pH 8, 0.5 mM EDTA ).

Comment: The Form IV, in a high-salt environment after alkali denaturation and neutralization, must be diluted again. This is necessary because the formamide-based protocol for separating the strands of Form IV, described in the next step, has only been reported in a low-salt setting. As you will see, if you watch Form IV – the Final Puzzle Piece, the behavior of Form IV is markedly affected by salt concentration, which must therefore be carefully controlled.
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(6) Remove the Form IV from dialysis. Mix one volume with 9 volumes of formamide. Incubate the mixture at 80º for 10 minutes.

Comment: Although formamide cannot, by itself, separate the strands of native circular DNA, it seems almost certain that it can separate the strands of Form IV. This is explained in Methods for Non-Destructively Separating or Reannealing the Strands of Circular Duplex DNA Chromosomes, available on this web site, or by clicking the above link. This is therefore the critical step in the non-destructive separation of the strands of the formerly-duplex chromosome, resulting in a nearly-pure population of single-stranded circular DNA.
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(7) The DNA can now be used directly, without further processing, as a hyperphase for electron microscopy, which will identify the product as intact, single-stranded circular DNA.

Comment: The art of distinguishing between single-stranded and double-stranded DNA by EM seems to be something of a "lost art". In the "olden days" (i.e., mid-late 20th century) it used to be done by complexing the SS DNA SS with single-stranded DNA binding proteins, such as certain capsid proteins from SS DNA viruses, which, paradoxically, actually makes the SS DNA look larger than DS DNA.

There may be newer and more efficient methods for the identification of SS DNA; methods that I do not know about. Be creative.