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

The reason you are having problems finding exact answers is that the information you are looking for is proprietary information meaning it is owned by the company that makes the kit. A patent search is the best way to find the answers you are looking for and still not infringe on anyone's intellectual property which would result in a major lawsuit.

However, as a scientist and knowing you are trying to bind DNA and separate (clean up other cell products from sample) one can make some speculations re stated kit components as you have attempted to do already.

Ethanol is a denaturing solvent w r t proteins (I agree).

Does the G-Cl enhance silica binding? I would have to say it is an essential component otherwise why would one want to add a naturally occurring base to their DNA sample and contaminate it with foreign matter. Unless of course, it is a monomer and is lost from the long-chain DNA product which the ethanol being an organic solvent can also help with. This is my reasoning. However, I am no expert in silica binding. Nevertheless, I have used it many times to separate an analyte using TLC plates and I can tell you that it is highly hydrophilic (water-loving) and a very soft, delicate material. So I can see one problem using this kit with a cell digest. Trying to keep the aqueous components from over-taking the silica. Obviously DNA is a biopolymer that has a sugar phosphate backbone but more importantly it has nitrogen containing bases that are not especially hydrophilic. Therefore, one must take advantage of the backbone to bind it to silica. This is a charged backbone since is based on phosphate and is polar because of the sugar residues. I see the G-Cl making a salt bridge with the internal N bases of the DNA sample to stabilize it for transfer to the silica but this is just my thoughts based on my 32 years of lab experience, knowledge of theory and problem-solving skills. Hope this helps! Alison D.