What are the roles of guanidine-HCl and ethanol in binding of DNA to silica?

Question

I'm trying to understand how exactly the binding to silica gel (in kits) step works and I cannot find any papers which provide an explanation of the physics or chemistry; especially on the way that Guanidine-HCl works.

So lets get them from the beginning.

First of all, we adjust DNA binding conditions by adding a buffer which includes Guanidine-HCl and ethanol and then we centrifuge our sample. I knew that Guanidine-HCl destroys the tertiary structure of proteins but somewhere I read that also it helps the DNA to bind to a silica column. I searched to find the way that it works but I couldn't find a paper. Also what is the role of ethanol in this step? Is it just to remove proteins and polysaccharides from the sample?

Answer 1

Guanidine HCL is a chaotropic salt. Chaotropic salts are critical for cell lysis and binding to the silica resin. Specifically, Chaotropes have two important roles in nucleic acid extraction

1. Destabilize hydrogen bonds, van der Waals forces and hydrophobic interactions.

   • Leading to destabilization of proteins (including nucleases).

     Macromolecular structure and function is dependent on the net effect of these forces (see protein folding), therefore it follows that an increase in chaotropic solutes in a biological system will denature macromolecules, reduce enzymatic activity and induce stress on a cell (i.e., a cell will have to synthesize stress protectants). Tertiary protein folding is dependent on hydrophobic forces from amino acids throughout the sequence of the protein. Chaotropic solutes decrease the net hydrophobic effect of hydrophobic regions because of a disordering of water molecules adjacent to the protein. This solubilises the hydrophobic region in the solution, thereby denaturing the protein. This is also directly applicable to the hydrophobic region in lipid bilayers; if a critical concentration of a chaotropic solute is reached (in the hydrophobic region of the bilayer) then membrane integrity will be compromised, and the cell will lyse. [Source]

2. Disrupt the association of nucleic acids with water.

   • Thereby providing optimal conditions for their transfer to silica.

     Salts can have chaotropic properties by shielding charges and preventing the stabilization of salt bridges. Hydrogen bonding is stronger in non-polar media, so salts, which increase the chemical polarity of the solvent, can also destabilize hydrogen bonding. Mechanistically this is because there are insufficient water molecules to effectively solvate the ions. This can result in ion-dipole interactions between the salts and hydrogen bonding species which are more favorable than normal hydrogen bonds. [Source]

Ethanol, like Guanidine HCL, is a chaotopic agent. Ethanol is added for 2 reasons:

1. Enhance and influence the binding of nucleic acids to the silica.

   • Interfere with non-covalent intramolecular forces as outlined above.

   • Reduce association of nucleic acids with water. From Melzak et al. (1996):

     Decreaing the water activity in solution through addition of either a chaotropic salt or an alcohol changes the helical structureof B-DNA either continuously to C-DNA or through a sharp cooperative transition to A-DNA (28). Either transition is accompanied by a decrease in solvent-accessible surface area.

2. The correct concentration allows for washing of the salts from the membrane. [Source].

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Citations

• Melzak et al. (1996) Driving forces for DNA adsorption to silica in perchlorate solutions. J. Colloid Interface Sci. 181: 635 - 644.