MadSci Network: Molecular Biology

Re: Lithium chloride (LiCl) and RNA purification?

Date: Sun Jul 30 18:28:06 2000
Posted By: Dr. James Kranz, Post-doc, Biochem & Biophys
Area of science: Molecular Biology
ID: 964677866.Mb

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

	(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.

Dr. James Kranz

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