Unraveling the Mysteries of DNA Extraction and Precipitation: A Practical Guide

[brovash]

I am trying to complete an assignment about DNA, and the following questions are nowhere to be found in my textbook/notes nor in any easy-to-understand journals! so any help would be greatly appreciated

- The difference between DNA and RNA that allows them to be separated...
I know that DNA has a deoxy portion on the ribose, and is double stranded instead of single, but is this what allows it to be soluble and separable in certain solvents that RNA cant? and if so how exactly?
- what base does to DNA and how it affects its viscosity if at all...does it base hydrolyze it or? this one's really confusing me
- what acid does to DNA and how it affects its viscosity if at all...again same as above...really confused
- DNAse affect on visocity of DNA ... this one I know that DNase cleaves and cuts the DNA into parts, and that it reduces its viscosity...but how? it is simply because the molecule is in many smaller parts so it can flow faster or something like that?
- how is DNA precipitated...is it like denatured with certain substances? or is there some sort of charge issue?

Of course I don't expect input on all of these, but any would be really helpful. Thanks!

 

[PuzzledMe]

Some of these questions are pretty interesting and I don't know either! I've found some leads on the difference between DNA and RNA. It has something to do with their relationship with Phenols and Phenols pH on extraction process.

The rest are deep, sorry I can't help :(

 

[UMich1344]

I also do not know many of the answers and am interested in what you find. Make sure to post your answers once you get them. It would make for a very interesting read.

 

[Spirochete]

A cop out answer is that you may not necesarrily have to chemically separate them using selective solvation (called elution). Remember that RNA molecules are generally composed of many fewer nucleotides than DNA molecules. This could be exploited by running the DNA/RNA mixture in an electrified agarose gel. The RNA will migrate much further toward the positive end.

This method is often sufficient if you are trying to isolate some DNA.

 

[Moonbear]

A cop out answer is that you may not necesarrily have to chemically separate them using selective solvation (called elution). Remember that RNA molecules are generally composed of many fewer nucleotides than DNA molecules. This could be exploited by running the DNA/RNA mixture in an electrified agarose gel. The RNA will migrate much further toward the positive end.

This method is often sufficient if you are trying to isolate some DNA.

Indeed, if you look at some of the commercially available kits for separating DNA, RNA and protein, and look at the methods used, you might get some hints.

 

[epenguin]

I'll start with your question about precipitation of DNA since that is on the one hand one of what Watson in 'The Double Helix' called 'the witchcraft-like techniques of the biochemist', on the other hand it is one of the nicest things to do in the lab. It precipitates in cold ethanol with an addition of also a % of isopropanol (don't ask!) but what you do is you mix the DNA (dissolved in water/buffer) and the alcohol while stirring with a glass rod. At a certain point you find clear or slightly whitish stuff is adhering to the rod it gets larger and eventually this viscous glassy stuff wrapped around the rod can just be lifted out of the solution, drained and redissoved or dried. A very nice thing to do!

Before that, to get more or less DNA and not much else there. You have to extract from some biological source, remove lipids which you do by extraction with phenol and/or chloroform, proteins whch you do with proteinases, which may also be used to disrupt the cells you are extracting from, and shaking with the phenol/choroform also denatures proteins which gather in the aqueous/organic interface so that gunge can be removed, at some point there is a high salt concentration which causes dissociation of proteins from the nucleic acids they are bound to. RNA is removed by ribonucleases. Here a trick is that there are RNAases that do not need metals, whereas most DNAases - present in the cells and that would start working as soon as you disrupted them - need metals like Zn2+ or Mg2+ which are inhibited by putting in a chelating agent.

Yeah, the viscosity of DNA is due to it being long sticky molecules, as it is cut to shorter its viscosity decreases.

RNA is much more sensitive to alkaline hydrolysis than DNA, because the 2'-OH participates in the hydrolysis with an intermediate 2'-3' cyclic phosphate diester.

You are right that the theoretical textbooks and Scientific American type journals do not tell you much about these aspects and you find them in practical manuals. DNA has to be extracted from different types of tissue for different purposes, so methods vary. A classic recipe was in Marmur, Jnl. of Molecular Biol. 3 208

Hope that helps but you need to see practically oriented books and I hope you can do that extraction/purification yourself once.