Okay, it's really cool. I did once get a mention with several sentences in Newsweek (with a very distressed Mickey Mouse on the cover) but this time I got several column inches. However, after I gave the phone interview I came down with a small case of the worries. What if I was misquoted? Worse, what if I was correctly quoted but pulled a Watson? Luckily, what made it in fails to induce embarrassment, though there are bits which I wish hadn't been left out.
The article is well worth reading (though it may become a pay article overnight; I forget the current policy). With luck the wire services & aggregators will pick up on it.
I think anyone interested in genetic testing, DIY-bio, or just science in general should skim the comments thread. There's a lot there to be worried about.
First, a running theme is a worry that Kay will blow up her block or such. Multiple posters, many claiming to work in labs. Now, as Kay's comment (which is nice and level-headed, as I would have expected) points out, she's not using anything liable to do anything like that. For the level of ethanol precipitation she's doing, a fifth of vodka would last quite a long time (an interesting experiment; I remember the Russians are said to have built lasers with the stuff).
A second class of fear is other sorts of toxins, primarily the spectre of ethidium bromide (a known carcinogen) as a DNA stain. There are other, much safer stains, and it turns out that's what's Kay is using.
Another general negative sentiment is that perhaps the city or her landlord should be (or might) shut this down. I'm no lawyer, but this certainly wasn't obviously prohibited by any of my lease agreements. Putting household cleaners in the public's hands (or solvents in the form of nail polish or paint removers) scares me far more than a little PCR.
One more sentiment worth noting: that this sort of thing should be done only in an official laboratory and that Kay shouldn't do this without getting a masters or Ph.D. first. I suspect that these posters aren't aware that many of the same techniques are available in the toy section of any Target or Wal-Mart. True, none of those offer PCR -- but they easily could. PCR can be run without any special gear, though it would be awfully tedious. They are probably also unaware of modern scientists who worked without Ph.D.s (e.g. Nobelist Gertrude Elion) or in home labs (e.g. Nobelist Rita Levi-Montalcini)
On the other end of things, some of the positive posters are a bit worrisome. One makes the quite apropos comparison of this to having a home darkroom, but gets their chemicals confused -- while the stop solution is indeed just acetic acid, the fixer is not "drinkable but dull" but rather cyanide-based (cyanide is a great remover of silver, which is the job of the fixer).
There are also a number of posters who suggest that this information might be used against her by an insurance company or that it would be illegal to withhold it from same. Whether this would be prohibited by GINA isn't considered; I'm guessing the poster's aren't familiar with it. Another poster relishes the idea that
Perhaps she objects to the greed of her peers at Harvard who are charging people for the opportunity to get similar bio data - See http://www.genomeweb.com/blog/round-100.-- which is bizarre, given that the very GenomeWeb article mentions that these tests are free to participants!
Regardless of how poorly informed or quick to leap to conclusions some of these folks are, this is indeed the landscape of public opinion, at least as plumbed by response to this article. It would suggest that there is a lot of educating to do & that it will be an uphill battle. To a lot of people, science means formal labs and formal training and labs mean dangerous chemicals that might explode.
Hi Keith,
ReplyDeleteTito Jankowski wrote to me about this project and I told him that the project wasn't very good scientifically.
Why?Because he said - and maybe Tito made a mistake in the description - that she was going to detect mutations by their failure to hybridize to the probe - without doing the normal (okay - normal, for me) things like including positive controls.
Either Tito doesn't describe things well, or something is missing.
I suggested RFLPs. Then, you can know if your PCR worked.
Did Tito just make a mistake in the description? What is Kay using as a positive control?
I don't -- I've let Kay know of my post & with luck she'll fill us in.
ReplyDeleteI'm using allele-specific PCR. The protocol is taken pretty much verbatim from Mullighan et al (1998).
ReplyDeleteThe idea is, if the SNP causes a mismatch at the 3' end of a PCR primer, this will cause the PCR reaction to fail. No PCR = no band. You can run a gel to check.
There are two canonical SNPs for hemochromatosis, at least for Euros like me, and they're close enough to hit in the same PCR reaction. So you need four primers - two for each SNP, one normal and one mutant. One SNP forward, the other reverse. Then you set up four PCR reactions to cover all the logical possibilities.
You're only going to get hits on one or two, so there's the others as negative controls. It's also prudent to run no-sample reactions, to make sure the hits are actually from you and not from your housemates. There's another positive control, to make sure the PCR worked - you can throw in another primer pair that's guaranteed to work. This protocol uses part of the HGH gene.
I'm not even doing ethanol extraction, just boiling with Chelex. I'm not sure what I could do to hurt someone with this protocol, short of dropping the thermocycler on their toes. (It's on the bottom shelf for a reason - that thing is a beast.)
Thanks Kay!
ReplyDeleteAre the SNPs you are looking at causative or simply tightly linked to the actual disease-causing variants? With your father it's of course known to be linked, but on your mom's side it wouldn't be so certain.
Kay, you said that " you can throw in another primer pair that's guaranteed to work."
ReplyDeleteThat would be a positive control for some elements of the PCR. It would tell you that your buffer, conditions, and enzyme were okay for one of the primer pairs.
However, it would not tell you anything about the other primer. If you have two different primers, they have different sequences and different melting temperatures. One primer does not suffice as a positive control for another.
You need to have the proper kinds of samples to ensure that all of your primers work.
BTW, the article Kay cites is in PubMed Central
ReplyDeleteYou need to have the proper kinds of samples to ensure that all of your primers work.Right. I'm hoping that using a validated set of primers (from the paper) will give me some degree of assurance, but ultimately the best check is to make sure the results come out correctly on known samples.
ReplyDeleteI'm a semi-known sample, assuming my paternity is what I think it is. And I'm going to become a fully known sample fairly soon - as soon as I get a job and insurance. And we'll see who else I can talk into this. Anyone of non-European ancestry could be a normal control, for instance.
Are the SNPs you are looking at causative or simply tightly linked to the actual disease-causing variants?The SNPs are causative. They're missense mutations in the HFE gene, which is involved in the negative feedback loop that tells the intestinal cells that iron stores are sufficient. Without that signal, the intestines continue taking up iron, which builds up and eventually causes organ failure.
Perhaps I'm missing the point, but IF that locus is known ... it's not *that* expensive to order a pile of primers and just sequence the region? Surely not *much* more expensive than ordering Taq, dNTPs, etc.
ReplyDeleteThat had occurred to me too & I'm guessing it's a matter of preference. The allele-specific PCR has the advantage it can all be done in a kitchen, whereas sequencing would require either (a) an expensive & cranky instrument (b) tricky & cranky manual equipment (and very challenging non-radioactive, non-toxic detection) or (c) shipping it out to a vendor. (3) is the obviously superior option, but not very DIY!
ReplyDeleteSequencing, of course, would find any novel variants that haven't been reported in the literature. Even if this locus has been hammered hard, I'd bet there are more disease-causing variants hiding amongst the 10B human chromosomes currently embedded in live humans.
Sequencing was an option, and I considered it. You'd only need two reads, one per SNP, though you could add on a few more for completeness. There are vendors that provide this service fairly cheaply. However, you'd still have to go through sample prep - you can't just scrape your cheek and stuff it into a sequencer. That would most likely mean PCR amplification of the relevant chunk of genome, then running a gel to confirm presence of correct band. At that point, you're not saving any work.
ReplyDeleteI also wanted to put together a protocol that could be real DIY - if you just want the answers, you can easily ship off a blood sample to one of hundreds of diagnostic labs, and they'll figure it out for you.
This is an education project. Nothing like squinting at a lousy gel to remind people what real science looks like - there's no Google button for the universe, but we're doing the best we can.
I brought the gel with me when I spoke at CodeCon. I think it blew a few minds. See that smudge up there? That's the bug in my family's source code. Yes, you can see it. Kind of. Hold it up to the light. (It was stained with methylene blue, so I didn't have to lug a homemade transilluminator through airport security.)