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Salting Out and Denaturing DNA

last edited by 7 years, 5 months ago

8.3.P5

Let's consider a (negatively charged) segment of double-helical DNA in an ionic solution consisting of equal numbers of positive and negative ions, as illustrated in the cartoon below (taken from Phillips, et al., Physical Biology of the Cell.)

A.  Why don't the positive ions attach all along the length of the DNA until the DNA is electrically neutral?  What, if anything, opposes that process?

B. The positive cloud of ions surrounding the negatively charged DNA extends a distance λD, the “Debye length.” Just outside the Debye length, the electric field magnitude is smaller than it would be if not for the positive ionic cloud.  "Salting Out" is the process by which pieces of DNA "clump together" and precipitate out of solution.  Does the presence of the ions in solution make it harder or easier for this clumping to occur (compared to the case of pure water)?

[NOTE:  In order for the DNA to actually clump up and precipitate out of solution, we often have to employ centrifugation (spinning the test tube containing the DNA around at a very fast rate).  Why do you think that is necessary?]

C. Discuss with your group how the Debye length would change with (1) temperature and (2) salt concentration, and give an argument as to why these dependencies are conceptually plausible.

D.  Using your answers from Parts B and C, decide whether the "salting out" of DNA from solution is more likely to occur as the temperature increases or decreases.  Decide whether the "salting out" of DNA is more likely to occur as the concentration of salt gets larger or smaller.

E.  Now let's zoom in a bit and look at the structure of a segment of DNA more closely.  One way to denature DNA (i.e., to break the double helix into two single strands) is by changing the ionic strength of the solution in which it exists.  DNA denatures quite easily when moved from a solution of considerable ionic strength (salt water, for example) to pure distilled water.  Why might it be much easier to denature DNA in pure water than it is in salt water?

Ben Geller 2/29/12