Coincident with the safety audit, there's been a bit of a discussion over at the Science Advisory Board about ethidium bromide, 'safer' alternatives and other ploys used by unscrupulous marketeers to get you to buy their company's product.
Ethidium bromide (EtBr) is a chemical that is used to 'stain' nucleic acid (DNA, RNA) so that we can actually see it in gels. Because EtBr allegedly comes up as a mutagen when assayed using the Ames Test (and google for it yourself), it's got quite the reputation for being one of the worst things in the mol biol lab. This is an ideal situation for the talentless hacks marketeers, because it means they can play on lab rats' fears to sell over-priced, under-performing crap.
It turns out that the whole argument against EtBr is so much elephant poo anyway. Perhaps the strongest evidence for the continued use of EtBr is that it has been used for years to treat sleeping sickness in African cattle. And if you do the sums, to get the EtBr dose equivalent that is used to treat one cow, you'd need to drink fifty thousand litres of gel staining solution.
Moreover, when a company decided to test their own EtBr substitute for toxicity and mutagenicity against the 'leading brand', they found that yes, their alternative was safer than the competition, but there was no evidence that it was any safer than EtBr itself:
In the graph, notice how SYBR Green — the 'leading brand' — goes up, which is bad, and then goes down, because the test cells are dying, which is very bad. EtBr ('EB') itself seems to be about as dangerous (i.e. not very) as EvaGreen, the company's chemical being tested. Notice that there is no dose-response effect. That is, in general, if something is dangerous, then the more you put into the system the more effect you get. Here, that does not happen for EB (or EvaGreen), which implies that any mutagenicity that is being detected is noise in the system. Indeed, I might suggest that the falling off in 'mutagenicity' in the three right-most EvaGreen columns is itself indicating a cytotoxic effect. Gosh.
And given that these alternatives are generally less sensitive than EtBr, which means you have to use more, EtBr begins to look like a clear winner.
Comments
I'd guess I'd better start drinking the stuff now.
What about that Sybr Gold stuff? It just *sounds* more dangerous than green.
Posted by: Whiffling and Ingesting | November 18, 2007 01:24 PM
umm, why don't you include the other figures from the study?
Sybr Green was only cytotoxic in one strain of Salmonella (It had no effect in the other one they tested).
Also, when the dyes were first mixed with liver enzyme extract (to mimic what happens if you actually get it into your body), EtBr was 7-1000 times more toxic than the other dyes.
Further, keeping cows alive a few more years longer before you send them to market is a little different than living a complete human life cancer-free.
I use EtBr all the time and have no plans to switch to an alternative dye. I think it is fine as long as you take the necessary precautions. However it is certainly not as innocuous as you make it out to be, and promulgating misleading information about its safety only makes it more dangerous as people will not treat it with the respect it deserves.
Posted by: pluto | November 19, 2007 04:02 AM
Thing is that everything is harmful, in sufficient quantities. There is a lot of paranoia about EtBr, which is a world away from 'respect'.
Posted by: bk | November 19, 2007 06:27 AM
Regardless of the realities of Ethidium Bromide teratogenicity (and by the way, I’m beginning to notice a trend wherein you seem to have unique insight into truth whereas the rest of the world are ignorant followers – this in itself is a form of paranoia, and I urge you to follow up with your self diagnosis of marginal Asperger’s Syndrome), the reality of working with ethidium bromide is that I have to manage an additional waste stream, which puts a considerable mental tax on the addle brained students in my lab, who can barely get their garbage into any waste bin, much less the appropriate one. For me, this is reason enough to embrace SYBR Green, which does not require any special warning labels, tea bags, or dedicated equipment.
One additional benefit is the ability to visually isolate bands while avoiding exposure of sometimes delicate DNA fragments to UV light. Yes, I know there are ways around this, but really there is no sin in utilizing the conveniences of advanced product development, even if it is ‘tainted’ by marketing ploys. Furthermore, while employing appropriate safety precautions should theoretically prevent a researcher from damaging themselves while hovering above a UV light box, desperately searchi8ng for invisible bands, I daresay every one of us has such an anecdote archived in their “oops” files.
And as far as decreased sensitivity, I have sent you evidence to the contrary under separate cover.
XOXO,
SD
Posted by: Sandiablo | November 19, 2007 11:09 AM
It's a fair cop, guv. My complete lack of social skills enables me to ask questions that other people don't. So rather than being paranoid, I see it as not taking any assumptions for granted.
Be that as it may, your contrary evidence is interesting — and counter to what others in this place have seen. Was that under UV or one of those fancy-pantsy dark light things? It must be nice to work in a modern, well-equipped and rich institute, instead of making do with what's there already, as we must. . . Maybe we had a dud batch of SYBR-safe?
Either way, there is a serious point half-made about waste disposal. You have this extra waste stream for EtBr (so do we), yet presumably the SYBR-Safe just gets thrown out in the normal trash? But if the evidence is that in the the amounts we use the S-S is more dangerous, then there is a serious problem here.
I'm not claiming special insight; I'm questioning assumptions.
Talking of which, another correspondent of mine disputes the dogma that shortwave UV does knacker your cloning. I have nothing other than anecdotal evidence that is relevant here, but I would be interested in other people's experiments.
Posted by: bk | November 19, 2007 11:57 AM
BioTechniques 21:898-903 (November 1996)
I was having dificulty cloning a EcoRI/NotI fragment when I came across this paper. I did a side by side experiment adding guanosine to my gel, and that baby slipped right in. Can't say for sure it was due to UV and/or EtBr damage to my ends, but it worked for me.
Posted by: SanDiablo | November 19, 2007 12:08 PM
Interesting paper. I'll talk about it in its own post. Wonder how it fits in with thymine dimerization?
Posted by: bk | November 19, 2007 12:11 PM