How ethanol precipitation works? - (Jul/06/2005 )
Can someone please explain to me chemically how this works?
when you add 100%ethanol in an aqueous solution, the solution become more than 80% ethanol in most cases. As DNA is soluble only in solutions up to 65% ethanol, it start forming small particules, but an anarchic auto repliment. The volumic mass of such particles is bigger than the one's of the solution, and centrifugation make the particles to pellet.
Why it works... (Frank, M. B. Ethanol Precipitation of Nucleic Acids. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/etohppt.html). 1997. Oklahoma City. Revision Date: October 2, 1997." Notes that appear below were taken from a discussion on this topic from one of the bionet newsgroups.)
A discussion on this topic was posted to the "bionet.molbio.methds-reagnts" bulletin board in July 1992. A thorough discussion was presented by Scott Keeney, of the Biochemistry and Molecular Biology Department, University of California Berkeley (scottk@nuclease.Berkeley.edu). I have shortened the opening questions and extracted his answer from the newsgroup and present them below for your convenience.
Q: What is the role of ethanol in precipitating DNA from aqueous solutions?
A: The purpose of adding salts is to neutralize the charge on the sugar-phosphate backbone of the DNA, but ethanol's task is a little more complex than "removing" the water. For a precipitation, you're interested in forming ion pairs between the polyanion (DNA) and the cation (Na+, Mg++, spermidine, protamine, etc). In dilute aqueous solution, DNA and counterions like Na+ and Mg++ are more or less in the free ion form rather than the ion pair form (that is, they are surrounded by one or more layers of water molecules).
Water has a high dielectric constant (e), which from Coulomb's Law tells us that the electrostatic force (F) between two ions of opposite charge is very low in water:
F = (Q1 * Q2)/(e * r2)
where Q is the charge on each ion and r is the distance between them. Adding organic solvent *decreases* the dielectric constant of the solution. As e goes down, F goes up and *BANG*, anion and cation form an ion pair and promptly swoon out of solution.
Q: Why does NaCl increase the stability of DNA duplexes, although you might expect salts to interfere with hydrogen bonds, rather than strengthen them?
A: The Na+ neutralizes the charge. Each strand of DNA has an enormous charge density (charge per unit volume), so the two strands tend to push each other apart. Cations added to the solution form a "cloud" of positive charges around the DNA. This cloud of counterions lowers the effective charge density and relieves the repulsion between the strands.
As for the effect of salt on hydrogen bonds, you have to realize that the hydrogen bonds formed between bases in duplex DNA contribute little to the stability of the duplex. For an interaction to stabilize the duplex, the interaction between bases must be stronger than the interaction of the bases with water (if bases are not paired with one another in a duplex, then they are surrounded by water). Hydrogen bonding between the amines, carbonyl oxygens, etc. of G-C or A-T is of the same energy (sometimes even less) than the hydrogen bonds these same groups would form with water if the DNA were single-stranded. (The H-bonds do contribute *something*: GC base pairs with three H-bonds are harder to melt than AT pairs with two.)
So, what drives DNA strands together? Entropy and enthalpy, of course. Entropy in the form of "hydrophobic" interactions between the bases (those big aromatic rings are quite hydrophobic, you know). Enthalpy in the form of favorable, stabilizing interactions between the pi electrons of the aromatic rings of bases as they stack on top of one another.
So, what do hydrogen bonds do, if they don't stabilize the duplex? They enforce the *specificity* of base pairing. Correct base pairing is nice, but doesn't add much. Incorrect base pairing, on the other hand, takes a lot away. Forcing unpaired H-bond donors and acceptors (i.e. hydrophilic groups) into a hydrophobic environment makes everybody unhappy.
“Ethanol or isopropanol or alcohols decrease the dielectric constant of the solution.
Water has a high dielectric constant (e), which from Coulomb's Law says that the electrostatic force (F) between two ions of opposite charge is very low in water:
F = (Q1 * Q2)/(e * r2)
where Q is the charge on each ion and r is the distance between them.
In an aqueous solution, DNA and counter ions like Na+ and Mg++ are more or less in the free ion form rather than the ion pair form i.e. they are surrounded by one or more layers of water molecules.
Adding organic solvent decreases the dielectric constant of the solution. As e goes down, F goes up and finally anion and cation form an ion pair and result in precipitation of DNA.” (http://micro.nwfsc.noaa.gov/forums/viewtopic.php?t=1623)