Month: July 2007

“Teh Deth Kitteh!”

Run! Itz teh Deth Kitteh!

The prestigious New England Journal of Medicine reports (Dosa, DM: New England Journal of Medicine; 2007; 357:4; pp 328-329 ) on the case of a single eukaryotic organism – a specimen of Felis catus – who is reported to identify People Who Are About To Die (insert ominous thundercrash here).

It is presented in a tone that is a mixture of “OOOo, spooky, mysterious!” and standard issue “Human Interest Story“, as though it was a baffling or unexplainable phenomenon. Honestly, didn’t modern science explain this long ago?

Obviously, Oscar the Cat is simply waiting around to devour the souls of the departed as they are exhaled on the last breath. Or as “Mike, the Mad Biologist” puts it:

Shouldn’t the situation be obvious? I mean, come on, did ALL of these journalists sleep through Biology 101? Even if they did, surely at least some of them own cats and already know about this….

There are, of course, numerous examples in the scientific literature documenting the tendency of the cat to steal the breath of the living. See, for example, Bener A, Galadari I, Naser KA.”Pets, allergy and respiratory symptoms in children living in a desert country”;Allergie et immunologie;1995 Jun;27(6):190-5…

Interestingly, as I was trying to find a more explicit reference in PubMed to this folk-belief, I ended up stumbling upon an article entitled “Micturition and the Soul” [Holstege G.”Micturition and the Soul.” J. Comp. Neurol. 2005 Dec 5;493(1):15-20.]. I love browsing databases of scientific papers. Where else could you go looking for a folk belief and find an article about the neurology of peein’?

I know I don’t normally discuss freakish, perverted Eukaryotes on this blog – hey, CHILDREN could be reading this! – but I found the article (and the responses to it) interesting, and it serves as filler until I can finish putting together my “Gram Stain Article To End All Gram Stain Articles” post.

Simplify, Simplify…

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.

Is GFP GRAS?

Woohoo!

One of my longstanding questions has been, is Green Fluorescent Protein safe to put in food?

I always figured that it SHOULD be – it comes from jellyfish, which I know people eat in some cultures (though I’m not sure if any of the ones eaten actually express GFP).

Well, it seems someone in 2003 did a proper test…Check it out!

Sure, this is still some way before the FDA declares it “Generally Regarded As Safe” but it’s one step closer.

Soon, my dream of genetically-engineered “Glogurt®” will become reality! AH, HA HA HA HA HA!

Okay then…

My summer classes are finally over. Got an “A” in immunology (go, me). Now I just need to make sure everything’s done next semester. I’ve already signed up for the last two Underwater-Basket-Weaving-type “General education” classes required at this college: Intro to Philosophy and “History of Western Art”. I also went ahead and signed up for Environmental Chemistry, too – it’s not required, but it’s one of the last “not required but useful if I have time for it” classes on my list.

Meanwhile – is it just me, or is DNA some obnoxiously fragile stuff when you don’t want it to be? Sure, leave a few flakes of skin or hair follicles at a crime scene and they’ll nail you weeks or months later, but try to “gel purify” some DNA and it just falls apart…

The samples from my last post, about the colony PCR of my Lactic Acid beer-bacteria, I cut the bright bands of presumably-16s rDNA out of the gel and ran them through one of those canned “gel purification kit” processes. Then I froze them until I had a chance to finish my classes and play with them.

Yes, I was wearing gloves. No, I didn’t lick the gel. I think I must have looked at them too closely or something and they just disintegrated out of spite. In any case, my attempt at a restriction enzyme digest turned up NOTHING (other than the “ladder” lanes) on the gel.

I’m beginning to really distrust canned kits. On the upside, that means I get to learn some more in the process of developing my own replacement protocols.

I will probably try re-amplifying DNA from the frozen samples and see if there’s anything at all left in there that can be saved. Otherwise, I’ll also check and see if the plates I made a few days ago still grew okay.

In other news – I’m toying with the idea of literally begging for my own microscope and home-microbiology lab equipment. As in, actually putting on a lab coat, taking an old hat, and sitting outside of scientific meetings and such with a cardboard sign saying “want my own microscope – please help”. Of course, I’d have to report any donations as “income” for tax purposes – I doubt they’d let me form a 501(c)(3) corporation dedicated to just buying me toystools for my own microbiological amusement.

I haven’t decided, but it’s under active consideration. It’d make for some interesting blogging (and I promise in return that I’d account on the blog for any money donated, and blog all uses of the equipment under Creative Commons terms so everyone can use it). It’d presumably take a while for this to get anywhere if it ever did – it seems it’ll cost about $400-$500 just for a (good) basic light microscope, plus another few hundred for a darkfield condenser and related upgrades. Plus, of course, me wanting to build some LED-based lighting for fluorescence microscopy ($500 canned commercial upgrade? Bah!). Incidentally, it seems Green Fluorescent Protein fluoresces best right around the wavelength of a typical, inexpensive, off-the-shelf ultraviolet LED…

And then of course I need a pressure cooker and one or more incubator setups and some petri dishes and trips to the grocery store for growth media and staining supplies and slides and… well, anyway, as much stuff as I can arrange to get. But the microscope is the one component that is unavoidably expensive.

Oh, yeah, and some space to keep cheese and beer culture organisms and such for later use…

Comments, anyone? Suggestions?