Re: [問題]請問酒精沉澱DMNA的原理是什麼
※ 引述《Helsinki (Helsinki)》之銘言：
: 要先加salt後再加95%酒精才行
: 沒salt的話DNA不會沉澱出來
: 因為salt靠正負電關係和DNA結合
: 加了酒精之後酒精搶不到DNA所以DNA可以沉澱出來嗎?
: 那如果要用isopropanol沉澱DNA的話沒加salt是不是也不會沉澱出來?
http://omrf.ouhsc.edu/~frank/ETOHPPT.html
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.
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