Difference between revisions of "Ethanol Precipitation"
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Revision as of 18:53, 29 July 2018
Oxygen has the right combination (of a number of protons with a collective positive charge of eight strongly attracting electrons, a small number of electrons, so that the electrons are highly attracted to the protons in the nucleus and not shielded by repelling each other, and is close to filling its outer orbital, so that two additional electrons beyond six in the outer orbital are very stable causing them to spend more time with the oxygen nucleus), so that it has a very high electronegativity (second only to flourine [1]) and tends to have a partial negative charge when covalently bonded to other elements.
This partial negative charge of oxygen results in a number of important phenomenon such as hydrogen bonding (in water partially positive hydrogen is attracted to partially negative oxygen of other molecules causing water to expand when it freezes contrary to the behavior of most molecules; this also causes the two strands of DNA to bind with each other) and being miscible with polar as opposed to nonpolar liquids.
Biological molecules have various levels of ability to remain in solution in water. This can be adjusted with alcohols, salts, silica, and resins that interact with the water and the biological molecules, at various levels to separate nucleic acids (DNA and RNA) from lipids, carbohydrates, proteins, and other biological molecules (metabolites and natural products) resulting from cell lysis.
The phosphate backbone of DNA is strongly charged because the oxygen pulls electrons away from the phosphate. This allows a large amount of DNA to remain in solution in pure water. However, the DNA molecule is also made up of sugars and bases that contribute less solubility to the entire molecule.
Salts are made up of ionic compounds that will generally dissolve in water and result in a negatively charged and positively charged components (ions). This does two things. The positive charged ions associate with the negative phosphates of a nucleic acid, acting to essentially cancel out part of their charge, and making the DNA (and RNA) less soluble in water. The ions also have a higher affinity for the charged water molecules and if present at high enough concentrations sequesters the water away from the DNA (see [2] and [3] for more extreme examples where alcohol can be separated from water using salt), also making the nucleic acids less soluble in water.
Alcohols have some partially polar properties (are hydrophilic and can dissolve in water) but are much less polar than pure water (the bound hydrocarbons shift the molecule more towards the hydrophobic direction). Nucleic acids are less soluble in a mixture of alcohol and water and insoluble in pure alcohol. Thus, increasing the alcohol content of a solution of water and DNA is also a method to precipitate the DNA out of solution.
This suggests purifying nucleic acids by combining a few steps. First mixing cell lysate in water with little to no salt or alcohol to dissolve the nucleic acids in solution. Spinning down or washing the insoluble biological molecules away from the DNA solution. Perhaps increasing the salt and/or alcohol concentration slightly to remove more biological molecules that are soluble in water but less so than DNA. Then increasing the concentration some more to precipitate out the DNA (but not so high that other highly soluble biological molecules are precipitated out of solution. This often results in a DNA pellet or DNA bound to a surface such as silica. This can be washed briefly with ethanol to remove other impurities. Then the resulting DNA is resuspended in a water solution that may or may not contain buffer depending on what it will be used for next. This solution will contain a mixture of DNA and RNA as well as some biological molecules. It should be stored frozen.
Links
https://openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids
https://bitesizebio.com/253/the-basics-how-ethanol-precipitation-of-dna-and-rna-works/
https://bitesizebio.com/2839/dna-precipitation-ethanol-vs-isopropanol/
