DNA seems to have two main threats to its well-being once it’s extracted and purified.
- Nucleases
- Spontaneous Hydrolysis by water
Nucleases are the big one that everyone seems to mention. The seem to be fairly sturdy enzymes, and they’re everywhere (including fingertips – hence the need to wear gloves whenever you get near DNA samples…), and they “eat” DNA rapidly. Theoretically, you can destroy the enzymes with enough heat, but you still need to worry about them getting in every time you pop open your sample to get some out.
Apparently, DNA even in pure water can tend to slowly fall apart spontaneously. It doesn’t happen very fast, but bit by bit, it can undo the links between the individual nucleotides.
A common way to try to deal with nucleases is to add EDTA to the solution. Nucleases need magnesium ions dissolved in the water to do their job, and EDTA tightly binds to magnesium (and calcium). The idea is to “use up” any stray magnesium ions in the solution so that even if nucleases get in, they’re inactive because they have no magnesium available. That’s why you see EDTA in the recipes for so many DNA-related solutions. Of course – EDTA doesn’t permanently bind up all the magnesium – there’s always a tiny fraction that stays in the solution. So, although EDTA can drastically slow down any nucleases, it won’t actually stop them.
There are also some interesting chemicals which can be added to destroy all proteins (including nuclease enzymes). Guanidine Thiocyanate is one rather nasty chemical that does this. 2-mercaptoethanol is another. Various other detergents like CTAB may also denature any proteins. Since they don’t harm the DNA in the process, you could keep the DNA sample dissolved in a solution with these chemicals…but then you can’t do PCR with the sample as it is, since the protein-denaturing chemicals will also destroy any enzymes that you WANT, like DNA Polymerase, when you try to mix it into your reaction.
I think the latter option will be great for collecting field samples (in fact, it’s papers specifically on the subject of preserving samples in the field with CTAB and Guanidine Thiocyanate based solutions that I’m adapting from), but isn’t going to be real useful once I’ve got my DNA relatively purified. What to do, what to do…
Actually, I think the answer’s simpler than I originally expected. I’ll just dry the purified DNA out. No water – no hydrolysis…and no nuclease activity, either.
I could actually just leave it as a dried pellet in the bottom of a microcentrifuge tube, but that leaves the problem of taking only a little bit of it for processing rather than taking the whole thing, and I want to avoid reconstituting it and re-drying it repeatedly. I think a variation of the “dry the DNA on a piece of paper” process will be in order – then I can just cut off a small strip of the paper to get a portion of the DNA. It appears that you can actually dunk the DNA-impregnated bit of paper right into whatever solution you’re using (like a buffered polymerase-and-primers solution for PCR) and go for it.
Among the several references I found on this, here are two:
Kawai J, Hayashizaki Y: “DNA Book”; Genome Res. 2003 13: 1488-1495
Burgoyne LA: U.S. Patent #5496562 “Solid medium and method for DNA storage” (1996); U.S. Patent and Trademark Office, Washington D.C.
It’s not clear to me what you’re worried about (I don’t know what your application is). If your DNA is reasonably clean, you can store it in the freezer or fridge for years without any loss. Hell, with plasmid DNA (if it’s clean) you can store it on your bench for a week, and it’ll be fine. DNA is not as fragile as some people think. Even RNA isn’t as fragile as folks suppose. To lose DNA from a sample, you have to be trying to lose it. (The amount you lose from spontaneous hydrolysis is effectively zero if you have any real amount of DNA). The only time I would worry is if I was starting with only a few ng. But if you have more than 100 ng of DNA, don’t worry.
The initial samples are actually quite dilute (~10-20ng/µl), but that’s not where I’m having trouble.
My real problem is that I’m doing method development, and I keep hitting problems and having to go back, tweak, and re-do steps, which means dipping back into previous steps’ samples.
One of these days I’ll do a real post on what I’m trying to accomplish, but in short, I’m taking a set of environmental samples off of graphite rods (by scraping), isolating the DNA from the samples (currently using a “soil extraction” kit, though I’d prefer to develop a fully-documented process of my own), running the samples through whole-genome amplification[1], amplifying the 16s rDNA sequences found therein, developing a clone library of the amplicons, and finally (when I finally reach this stage) getting sequences and hopefully observing differences between the samples.
Right now, it’s the 16s amplification that’s giving me issues – the initial amplification of bacterial (but presumably not archael) sequences initially works fine, but I always seem to lose the samples during purification.
I’m assuming I’m getting some kind of contamination, bringing in a little nuclease and destroying everything.
As a result, I’m trying to come up with a good way to both purify the 16s PCR product AND keep it in a stable, ready-to-use condition over long periods of time and repeated access.
I’ve been told that I should just make sure I go through the whole process from amplification to clone-library-starting in a single day, as though it were perfectly normal for things to be so brittle. To me, though, this doesn’t mean “I’m not rushing through this fast enough”, it means “this process is not sufficiently robust”. I’m trying to fix that.
There are other issues too (I also want to devise a multiplex PCR process so that I can get archael sequences, if present, and possibly use a nested PCR process for “purification” rather than one that involves directly handling the DNA in the open air and allowing it to touch multiple surfaces (i.e. “gel purification”).
[1] Wu L, Liu X, Schadt CW, Zhou J. “Microarray-based analysis of subnanogram quantities of microbial community DNAs by using whole-community genome amplification.” Appl Environ Microbiol. 2006 Jul;72(7):4931-41. (I’m not doing microarray analysis nor am I using the additional performance-boosting SSB or spermidine described by the authors, but otherwise it’s the same technique for a similar purpose.)