A team from Tokyo Tech have just published about a new, leak-free and easy to use method for amplifying DNA at 37°C! While the biologists who follow me are already getting excited, that might take some explaining for the laypeople, so here, let’s congregate in this corner to discuss DNA amplification. Comfy? Got your beverage of choice? Right!
What does DNA amplification mean?
When you’ve got a DNA sample, it’s usually not that big. DNA amplification takes a small amount of DNA — the DNA found in the cells taken by a cheek swab, for example — and multiplies it so there are millions of copies.
Right, but what is that useful for?
All kinds of things! If you want to compare two DNA sequences for any reason, you need to multiply the samples in order to compare them. If you need to know whether a particular gene is present, and you know how long it is, you can amplify DNA to create a sample you can then run through a gel, separating the contents out by length. This creates a band in the gel, and you can see whether there’s a band present at the right spot to be a gene of the length you’re looking for. DNA amplification techniques have let us analyse the mitochondrial DNA of Neanderthals, figure out who people’s parents are, test for disease, and explore all kinds of things about how cells work.
But we can do that already?
Yep! We have a method called PCR, which stands for “polymerase chain reaction”. It is a formidably smart way of multiplying DNA to the levels that are useful for lab tests. DNA polymerase is an enzyme that synthesises new DNA from existing strands. Every time a cell divides, DNA polymerase is right there. Within your cells, a helicase enzyme unwraps the two strands of DNA from one another, and then a polymerase links up with one of the strands.
A strand of DNA is made of four bases: adenine, thymine, guanine and cytosine. Adenine only ever bonds with thymine, and guanine only ever bonds with cytosine. So when you have the double helix of DNA, the two strands aren’t actually identical: one will always have an adenine where the other side has a thymine, and the same with guanine and cytosine. That means that by knowing one strand, you can create the other, and that’s what DNA polymerase does. It links up with a strand, and creates the complementary strand.
In a test tube, it’s a little more difficult. The strands of DNA are typically separating by heating them up… and if you know anything about enzymes, you probably know that heat typically “denatures” them — twists them out of shape and makes them unusable. Early PCR actually got round that by manually adding DNA polymerase after each heating cycle, but given a typical PCR reaction goes for 30-40 heat cycles, that’s a little bit tedious! But biology often has the answer, and the answer was a heat-loving bacterium with DNA polymerase that stays stable up to the 96°C temps that are used to make the DNA strands separate, and works at around 72°C. Much less heating and cooling is needed because of Thermus aquaticus — thank goodness for bacteria! Each cycle of PCR doubles the sample, so if you start with 10 DNA molecules, after one cycle you have 20. Then 40. 80. 160… You get the gist!
There’s one more catch, though, while I’m at it. DNA polymerase doesn’t know where to start. For that, you need to add primers to the mix. These primers need to bind to the DNA strand to show the polymerase where to bind and start copying. By choosing the right primer, you can choose where it binds to the DNA, and exactly what stretch of DNA you want to copy. This adds an extra cooling step to the process, because primers bind to the DNA at 68°C… and then the polymerase works best at 72°C. The temperature has to cycle up to 96°C, down to 68°C, back up to 72°C, and then do that again and again and again to amplify enough DNA to be useful.
So yes. We can amplify DNA already. But all of this still involves costly equipment.
What does leak-free mean, though?
“Leak” amplification is a problem with PCR whereby the DNA that’s amplified isn’t always what you intended. This can lead to false positives — a problem that’s particularly important when you’re diagnosing disease, or checking paternity, or identifying someone from traces left at a crime scene.
So what’s the new method all about?
The new method is called L-TEAM, which stands for Low-Temperature Amplification. The Tokyo Tech team are not only using a new method that works at 37°C, they’re also adding stabilising molecules that are radically reducing the amount of leak amplification.
Sooo… this is something to be excited about?
Sounds like it! All this said, I haven’t read the paper itself yet (hey, I was busy writing this blog post!). On the surface, it sounds pretty miraculous. I’ve done PCR, actually; it’s mostly automated now, but I swooned a little at hearing the price of the equipment, and it’s a pretty unforgiving process, for all that I think it’s immensely clever. If we’ve got a viable, simple alternative to it with fewer downsides, and particularly with cheaper equipment, labs all over the world are going to be overjoyed.
Either way, now you know about PCR [if you didn’t already], and if that doesn’t make the world a little bit cooler for you, I don’t know what to say to you. Maybe we shouldn’t be hanging out?
(I’m kidding. Mostly.)