|MadSci Network: Molecular Biology|
Dear "h5nl", The most widely use method of recovering DNA and RNA from solutions is through precipitation with ethanol, followed by resuspending the precipitated material in an aqueous solution. More specifically, the precipitate of a particular nucleic acid is formed in the presence of moderate amounts of monovalent cations, which act to shield the poly-anionic phosphate backbone of the nucleic acid in the compact precipitate, in concert with the dehydrating effect of added ethanol. These combine to form a multimeric aggregate, which is recovered by centrifuging the sample, thereby collecting all recoverable material in a single pellet, and is lastly resuspended in water. The efficacy of ethanol/H2O mixtures at precipitating nucleic acids is a function of three major variables: (1) the type and concentration of monovalent cations used in the precipitation. (2) the temperature at which the precipitate is allowed to form. (3) the time and speed of centrifugation The latter two factors are typically thought to affect the yield of recovered material, though the temperature at which one performs the precipitation is less important than is the time and speed at which the material is recovered. However, the first variable, the choice of the monovalent cation, can be a significant factor in exactly which type of nucleic acid is recovered. Without belaboring the detailed mechanism that drives precipitation, it is important to note that, in the case of nucleic acids, it is energetically unfavorable for individual molecules to form a compact mass. The array of negatively charged phosphates on the backbone of DNA and RNA tend to force other molecules away. However, in the presence of cations (such as Na+, K+, Mg2+, etc.), the phosphates on the backbone of the nucleic acid will be largely neutralized through ionic interactions with the solute. In terms of precipitation, the highly charged DNA or RNA acts as though it has a net neutral charge, effectively removing the charge-repulsion that would normally force them apart. The ethanol one adds drives the precipitation through dehydrating the surface of the nucleic acids; the cations facilitate the tendency of these dehydrated surfaces coming together. It's a little more complicated than that (the choice of anion has an affect on this process as well), but that covers the main points. The reason people use DIFFERENT types of cations is that they are more or less effective at charge neutralization, and therefore differ in their ability to effectively precipitate nucleic acids in the presence of ethanol. What that means to researches is one can selectively recover molecules of different sizes. For example, if ammonium acetate is used, one recovers both DNA/RNA molecules as well as dNTP's/NTP's (the individual building blocks of DNA or RNA, respectively). However, if one uses lithium chloride, one typically recovers large DNA or RNA molecules, but not small fragments or the NTP's. It's possible that in an RNA prep, the use of LiCl in a recovery step may be used to selectively recover mRNA or rRNA, without also recovering tRNA's (You'd have to do additional research to be sure, but that's one possibility). Also, it is known that LiCl is very soluble in ethanolic solutions and is typically not co-precipitated with the nucleic acid. This is not true of ammonium acetate or sodium acetate, which form a substantial weight fraction of the pelleted material; it is often the case that subsequent de-salting techniques must be used to eliminate these ions in the recovered material. In summary, the effects of LiCl on the recovery of nucleic acids should work identically for DNA or RNA of similar sizes. The use of LiCl over other added salts in ethanol precipitations is typically done as a means of selectively recovering larger molecules, without co-precipitation of smaller nucleic acid fragments or NTP's. For more specific information (such as appropriate concentrations, etc.), one place to start is "Molecular Cloning" by Sambrook, Fritsch, and Maniatis, Cold Spring Laboratory Press. I hope this helps. Regards, Dr. James Kranz
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