Figure 1: Free radical polymerization converts acrylamide into linear polyacrylamide chains of various length. The acrylamide unit in polyacrylamide is depicted at the right.
Historically ethanol precipitation was used as a method for the concentration of biologically active nucleic acids as early as the 1930s. Ethanol precipitation is now a standard method for precipitation of DNA out of a solution for removing salts and for resuspension of the precipitated DNA in a different buffer. Both ethanol and isopropanol allow precipitation of nucleic acid fragment out of solution. However, ethanol is usually the preferred choice.Ethanol precipitation is also used to concentrate DNA, RNA, and polysaccharides, for example pectin, from aqueous solutions. Alcohol precipitation methods allow recovery of DNA and RNA of high purity form biological samples. Protocols using alcoholic precipitation steps are now commonly used in diagnostics and protocols in molecular biology with the need for highly purified DNA or RNA. However, for efficient precipitation and recoveries with high yields, several vital parameters such as salt, alcohol, or carrier choice have to be considered.
Thirty years ago, Gaillard and Straus showed that linear polyacrylamide used as a neutral carrier allows precipitating picogram amounts of nucleic acids with ethanol. Chemically synthesized linear acrylamide, free of nuclease contamination, eliminates the risk of trace contamination introduced by the precipitation process. Polyacrylamides exhibit strong hydrogen bonding and water solubility resulting in a variety of uses in diverse industries.
Linear polyacrylamide also allows precipitation of RNA with ethanol or precipitation of proteins with cold acetone. Linear polyacrylamide does not interfere with spectrophotometric readings at 260nm and 280nm. Hence ethanol is the best choice for DNA and RNA precipitation.
Cations in salts neutralize the negative charge of the DNA phosphate backbone. Microcentrifuge tubes, in combination with a microcentrifuge, allow efficient precipitation of DNA or RNA with ethanol.
How does ethanol precipitate oligonucleotides?
DNA and RNA oligonucleotides have an exposed backbone of negatively charged phosphate residues that make these molecules highly polar. In aqueous solutions, charged residues attracted hydration shells of water molecules that suppress the binding of positively charged ions to DNA molecules. Ethanol disrupts DNA and RNA's hydration shell, allowing unshielded phosphate residues to form ionic bonds with cations in the solvent. At a concentration of 70% ethanol in the solution in the presence of 300 mM sodium cations reduces repulsive forces between the polynucleotide chains such that DNA and RNA precipitates. Ethanol precipitation occurs when enough cations are present to neutralize the charge on the exposed phosphate residues. Isopropanol is less polar than ethanol and has a higher propensity to precipitate salts and antibiotics as well. Table 1 shows the most used cations.
How can DNA and RNA precipitates be dissolved?
Often DNA and RNA precipitates recovered after a precipitation step, for example, using ethanol, are dried under vacuum before redissolving.
However, it is best to modify this practice because of the following reasons:
(i) Desiccated pellets of DNA and RNA dissolve slowly and inefficiently.
(ii) Small fragments of double-stranded DNA (<400 base pairs) become denatured after drying. Most likely as a result
of losing the stabilizing shell of bound water molecules.
Therefore, the best practice is to remove ethanol from the nucleic acid pellet and the tube's walls by gentle aspiration followed by storage of the open tube on the bench for approximately 15 minutes to enable evaporation of most of the residual ethanol. The resulting damp pellet can then be dissolved rapidly and completely in the buffer needed for the next experimental step. However, for quantitative removal of the ethanol, the open tube with the redissolved DNA or RNA can be incubated for 2 to 3 minutes at 45°C in a heating block to allow any traces of ethanol to evaporate.
Please note, after centrifugation using an angle-head rotor, precipitated oligonucleotides are not all found at the bottom of the tube. Approximately around 40% of the precipitated oligonucleotides are present on the wall of microcentrifuge tubes. Therefore, to achieve maximal recovery, use a pipette to move an aliquot of solvent over the surface with a disposable pipette tip.
For radioactive samples, check that no detectable radioactivity remains in the tube after removing the dissolved oligonucleotides.
To conclude, DNA and RNA ethanol precipitates can be redissolved quite easily in buffers of low ionic strength, such as in TE buffer at pH 8.0. However, when buffers containing MgCl2 or greater than 100 mM NaCl are added directly to the pellet, difficulties in the pellet's quantitative redissolving can arise. Hence, it is preferable to first dissolve the pellet in a small volume of low-ionic-strength buffer and slowly adjust the buffer's composition to the final buffer composition.
Sometimes, if the sample pellet does not dissolve easily in a small volume, a second precipitation step using ethanol is needed. This second step may help eliminate additional salts or other components that can prevent the oligonucleotides' dissolution.
Reference
Clerget G, Bourguignon-Igel V, Rederstorff M. Alcoholic precipitation of small non-coding RNAs. Methods in Molecular Biology (Clifton, N.J.). 2015 ;1296:11-16. [Europe PMC]
Gaillard, C. and Strauss, F.; Ethanol precipitation of DNA with linear polyacrylamide carrier. (1990) Nucleic Acids Res. 18, 2, 378. [PDF]
Green and Sambrook; Molecular Cloning. A Laboratory Manual. 4th Edition. Cold Spring Harbor Laboratory Press. 2012. pp. 21-27. [Book]
Also review "Development of a simple RNA extrection method ...... "
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