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Old 02-22-2012, 10:10 AM   #1
ALBraun
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Default AMPure XP Beads Size Selection Chemistry

Hello!

This question might be incredibly obvious but I have been trying to understand the chemistry behind the size selection using AMPure XP Beads to no avail. I don't understand how varying salt and PEG concentration can allow for selective binding of DNA based on the size or length of the fragment.

I was hoping that someone could explain this to me or point me to a good reference that explains the underlying mechanism behind DNA carboxyl binding size selection?

Thank you!

Lex
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Old 02-22-2012, 10:31 AM   #2
ECO
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It's not "binding" but precipitation. The surface of the beads only serves to nucleate precipitation.

Not in a position to add more now, but I have never seen a definitive reference that explains the precipitation chemistry. PEG/NaCl removes enough water to precipitate DNA...higher levels are required to precipitate smaller fragments. Don't add enough, small fragments don't precipitate.**


**WARNING: Molecular biologist talking solution chemistry. Use caution.
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Old 02-22-2012, 10:33 AM   #3
pmiguel
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I don't know the full detail, but you have two processes:

(1) PEG/High salt precipitates polynucleotides. The higher the MW of the polynucleotide the more efficiently it precipitates.
I generally think of precipitations of this sort being the result of the PEG occupying some fraction of the water molecules that normally would be solubilizing the polynucleotides. The longer the polynucleotide, the more water atoms need to be available to keep it in solution.
We used to do hard (microfuge) spins to collect the precipitated material.

(2) The beads bind polynucleotides via their charge. I presume this interaction is not enough by itself to pull the polynucleotides out of solution. But in the presence of precipitate they gather appropriately charged molecules to their surface.

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Old 02-22-2012, 11:06 AM   #4
pmiguel
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Quote:
Originally Posted by ECO View Post
It's not "binding" but precipitation. The surface of the beads only serves to nucleate precipitation.
Hawkins, T.L., et al. (1994). DNA purification and isolation using a solid-phase. Nucl Acid Res 22(21):4543-4. looks like the original "SPRI" paper.

Here is the comment about carboxylated beads from that paper.

Quote:
We have noted that under conditions of high polyethylene
glycol (PEG) and salt concentration (10% PEG 8000 and 1.25
M NaCl final concentrations) (6), DNA would bind to the surface
of carboxyl coated magnetic particles. Once bound, the DNAbead
complex could be extensively washed and finally eluted in
water to yield purified DNA.
Ref 6 above is Lis,J. (1980) Methods Enzymol. 65, 347-353. and is about PEG precipitation of DNA but no mention of carboxylated beads that I noticed.

The word "bind" above suggests more than a nucleation site to me. But because no mechanism is given for the binding, I have nothing to dispute a "nucleation only" hypothesis.

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