PCR: when you need to find out who the daddy is.

Aug 31 2010 Published by under Basic Science Posts, Neuroscience, Uncategorized

You all may remember when a certain political candidate made a comment concerning fruit fly research. It seemed like half the world reacted with "yeah, fruit fly research is dumb! Let's cure cancer!" while the other half went "hey, I think that's probably important..." and tried to come up with examples.

And while we've obviously moved on (at least temporarily) from such nonsense, Sci still thinks about it from time to time. Because it just makes it obvious how incredibly important it is that we scientists let the world know what we are doing, and what it all MEANS. How their tax dollars are being spent. What progress we are making. And to let people know that the science of curing cancer, or depression, or alcoholism, or Alzheimer's, or anything else, is about FAR more than just looking at a disease and testing potential drugs.

Because to look at a disease, and to test drugs, and to emulate a disease, you need techniques. You need ways to come up with drug targets, you need ways to understand what the drug is doing and HOW it's working. You need to assess side effects. And even before you get to drugs, you need to understand what the disease itself does, how it works, and what exactly is going wrong.

And to develop those techniques, you need research. And you never know what research is going to come up with something amazing.

And so, for today's example, Sci would like to tell (in a really, really basic way) about PCR, the Polymerase Chain Reaction.


(Who's your daddy?)

To do this, let's start with some DNA.


(That's some pretty stuff right there)

So DNA, aka deoxyribonucleic acid, is the stuff of life. You all probably know that DNA makes up genes, genes can make proteins, and proteins make up everything else. DNA is a very big deal, because the changes in our genes can have VAST changes in how we turn out, and in how we respond to things. Mutations in some genes produce cancer. Mutations in others cause albinism. And in some cases, even a single mutation is enough to ensure a zygote never even makes it off the ground.

There are a lot of diseases out there involving mutations in genes, from small mutations to very wide swaths of changes. The issue is, how do you DETECT these changes? You've got a boatload of DNA, and some of these changes are very tiny. So a lot of times, you need to make MORE DNA from the DNA that you have, creating enough of it so that you can analyze it and get a result that's believable.

Now used to be, you'd just have to culture a bunch of cells to do that. But then, in 1983, Kary Mullis came up with an idea to amplify DNA without using cells.

Here's how it works:

Take your DNA. Heat it up to 95 degrees Celsius, right below the boiling point of water. The DNA strand under that kind of heat will split, like so:

Then, you've got two stands, one going one direction (5' to 3') and the other going the other direction (3' to 5'). Now cool it down to about 50-60 degrees Celsius.

This allows a primer to bind. A primer is a bit of single strand RNA that binds to the DNA in a specific spot, creating a bit of DNA, which allows your polymerase to bind. A polymerase is an enzyme (a type of protein) that reads the single strand of DNA, and uses that to make another strand that is complementary to it. You use a primer for your 5' to 3' strand, and another for your 3' to 5' strand. The polymerases will bind to each strand and make a complementary strand to it by attaching the matching bases on the other side (obviously you put in enough bases for this), taking the two split strands of single DNA and making them into two DOUBLE DNA strands.

You'll notice you heat it up to 72 degrees for this step, which is the best temperature for the DNA polymerse to work at.

So now you've got two strands of DNA. Heat it up to 95 degrees again. Now BOTH strands will separate, and when you cool it down, all FOUR single strands will get primer on them, and all FOUR will be replicated.

1-2-4-16...

So as you run more cycles, the DNA will increase exponentially, until you have enough. Obviously this depends on a lot more things like whether you have enough nucleotides (the little chemicals that make up DNA), and the chemicals your DNA is bathed in. Other than that, it's really not that hard. We now have machines that can do this whole thing with a little prep in under two hours.

But here's the thing.

This should NOT BE POSSIBLE.

For a very simple reason. 95 degrees is really really hot. I mean, not boiling, but hot. My molecular biology teacher told it like this: put an egg in 95 degree water for 10 minutes. Sure the DNA will denature. But you'll also have a boiled egg. Because the PROTEINS will denature TOO. Proteins like DNA Polymerase.

Ooops. Polymerase is a little important here, you really kind of need that. You may recall up there that I mentioned that DNA polymerase is what binds after your primer and what forms the complimentary DNA strand. Without that, you aren't going anywhere.

So at first, you'd have to wait to add the polymerase in until after the hot cycle was done, but BEFORE the DNA had glued itself back together. Difficult, and slow. You'd have to do it for EVERY cycle. The average number of cycles you need before you can detect something is at least 40. You can see how this might be annoying.

And now enter the Taq polymerase. The Taq polymerase is a version of DNA polymerase that is found in bacteria (Thermus aquaticus) living in hot springs in places like Yellowstone. Places where you have to be REALLY tough to live. Not only do you have to be tough, your PROTEINS have to be tough. These hot springs are around 80 degrees Celsius. And so these bacteria have special proteins that are HEAT RESISTANT. They don't break down in the heat like our proteins would.

And when you use the Taq polymerase from this bacteria in a PCR reaction...well the thing can run forever! And FAST. You don't have to worry about adding polymerase every step, as long as you have enough nucleotides and primer, you'll run for ages, because these special heat resistant polymerases don't denature at the 05 degree step! They can keep chugging along when they cool down, just as usual.

Mullis first applied the use of the Taq Polymerase to PCR. He won the Nobel Prize.

And now. Let's pause and say incredulously "WHO THE HECK would spend TAX MONEY on little bacteria in HOT SPRINGS!! That's not going to help anything!!"

And now let's see what PCR can do:

1) Diagnosing cancers like leukemia
2) Detecting bacteria and viruses in samples from sick people, sometimes before they develop symptoms
3) Diagnosing genetic diseases and mutation
4) Paternity Testing
5) Forensics

And a lot more than that. PCR has been essential to looking at genes (and thus looking for cures for): cancer, Alzheimer's, Huntington's, autism, depression, anxiety, drug addiction, alcoholism, heart disease, thyroid disorders, diabetes, etc, etc. I could go on.

Entire BOOKS are now written on using PCR in human disease.

And if weren't for a guy studying bacteria in a hot spring, medicine would not be where it is now. I'm sure Dr. Brock never really thought about the potential applications until they happened. But I hope he's very pleased.

And that's the thing. You NEVER KNOW where the next breakthrough will come from. No well performed, innovative, original research is wasted. There could be a new drug for cancer discovered by a botanist (like Taxol, a very important chemotherapy drug in breast cancer, which is from a Yew tree. As far as I know, this was the first time the Yew tree had been considered for this purpose or any medicinal purpose, and it was part of a large screen of similar plants). There could be new chemicals for new techniques discovered from those tube worms living deep in the ocean. And even if the research that's coming out now on these things doesn't look like it will lead to something like this, be patient. Sometimes it takes some time to see the possibilities.

9 responses so far

  • Marius says:

    Last year I did a vision experiment on the street, to see where people look when they walk in a real environment (as opposed to where they look when watching pictures on a monitor in our lab). And people asked: "What's this for?" "Well," I said, "we want to know where people actually look in real life." "Yeah," was the answer, "but what's it for? What's the goal?"

    Who knows, perhaps this can be used in robots on rescue missions or for people with visual disorders, or maybe to design better streets where people don't fall as often. Right now, we don't have a clue.

  • Jill says:

    Thank you for doing these basic lessons. I've been enjoying reading them! And, yes, we need the scientists to tell the world what they are doing and teach them to appreciate basic science for what it is.

  • becca says:

    How come the DNA of Thermus aquaticus doesn't melt? THAT's the part that should be unpossible.

    • proflikesubstance says:

      High G/C content (almost 70%) and accessory proteins to stabilize the DNA. The adaptations to the environment are pretty cool.

  • A point to consider about the fruit fly research being done in France was that it was being conducted there to protect olive crops here in the United States.

    May as well do the research where the pest is endemic, rather than emerging. Makes perfect sense to me.

  • jgill says:

    Love this post. I think it's really important that we draw lines back to the foundations that we are building on a daily basis. Without this, we have a harder time inspiring future scientists, current scientists, politicians and tax-payers. Even though I work around science everyday, I often forget about the bigger picture and this type of exercise brings it into focus. Thx!

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