DNA's HyperChromic Effect

Detecting DNA's HyperChromic Effect
--- A Project ---

Carl W. Vermeulen, PhD

Williamsburg, Virginia

DNA's hyperchromic effect means that ssDNA absorbs more UV than does dsDNA).

An insect, which can see in the UV range,* would see the hyperchromic effect something like that shown to the right. At first the DNA solution is only a little violet. If it is boiled and then slowly cooled, it ends up a little more violet than it started, but if it is rapidly cooled it becomes most violet. The reason that this happens is that in dsDNA the pi-electrons in the aromatic rings are more constrained because the H-bonded rings are in sandwich layers - overlapping with each other. But if the H-bonds are "boiled" away, the sandwich no longer exists and the pi-electrons are more free to move into different energy levels and thus able to absorb more UV energy.

The cost of a UV-spectrophotometer is in the thousands of dollars, and the cost of SYBR Green-1 is beyond the pockets of most school systems. Thus another way must be developed. Most high schools have a newspaper, and having such, there are usually a few students who act as photographers, and sometimes they even develop their own photos. Thus doing simple photography should not be out of the question in most school systems.

All that are needed for this demonstration of the hyperchromic effect are:

  1. a UV-lamp that emits "short wave" UV
  2. Some microscope slides and long coverslips made of a UV transparent plastic - often these are sold as "disposable" and thus should be even less expensive than glass slides.
  3. Some wires of different guages (needles perhaps) to be used as supports.
  4. Some UV-sensitive photographic print paper (most B/W print paper is UV-sensitive) and the proper developers.
  5. A dark room with safe-light so the experimenters can see what they are doing.
  6. UV-opaque safety goggles.

I would think that most schools would have all of the above except, perhaps, the plastic slides/cover slips.

In essence, the dsDNA solution to be tested would be placed as a "wedge" between the slide and coverslip which is supported at one end. Thus the depth of the solution varies between zero and the width of the support. In the dark, a piece of the photo print paper is set on the desktop, and this setup is placed on top of the paper. A piece of thick black paper is placed between the "wedge" and the UV lamp.

Initially the UV lamp is "off". Once the piece of heavy black paper is between the UV lamp and the "wedge"; the lamp is turned on. Exposure time is determined by how long the black paper is manually removed from the intervening space (a common practice used in photo labs). ((This technique is used here because the UV lamps do not ignite instantaneously, and so electric timers are not useful.))

Then the wedge of dsDNA can be substituted with another portion of that solution that has been brought to a boil and quickly cooled. A similar exposure is made on print paper, and then the two papers can be developed. It should be remembered that the more exposed the paper is to UV, the darker it will be. Thus we should expect that the boiled DNA (ssDNA) should absorb UV better and thus the paper beneath should be less exposed - lighter.

And if you were an insect you could have seen that these wedges were different even without the UV light!

* You will have noticed that most white flowers are pollinated by insects, while red, yellow and blue flowers tend to attract birds. White flowers are not really white: they are in fact ultraviolet in color - a set of colors that neither we nor the birds can see. The astute observer may also have noticed large conspicuous spiders nesting in the center of certain white flowers. You might have wondered how any bug could overlook such a danger to life and limb, but while the spider is conspicuous to our eyes, it is not to insects' eyes.