The curious chemistry of gene sequencing

Each bit of DNA that leaves the UK Biobank over the next two years will head to either Iceland, to the company deCODE, or to the Wellcome Sanger Institute in Cambridge. But both places will sequence the genes of their samples in the same way - using tech from sequencing company Illumina. If you've listened to the last couple episodes, you heard how their machines can do a whole genome in forty hours. But we didn’t cover a crucial part: the chemistry, how the machine can look at only one letter at a time. Phil Sansom went to visit Vince Smith, who leads their chemical team...

Vince - We sequence the DNA inside something called a flow cell, which I have in my hand here. Just a couple of pieces of glass glued together with a layer in between them, and there's a chamber between those two pieces of glass.

Phil - It’s probably a lot more complicated than it looks, right?

Vince - Yeah, yeah, superficially it looks like a relatively small piece of glass. It's got a slightly rainbow colour, there's light refracting through it.

Phil - Yeah, it does.

Vince - And that's because of these very tiny patches that we load the DNA molecules into, billions of very tiny cylindrical wells - they're just a few hundred nanometres across.

Phil - Where does the DNA go on that?

Vince - Inside a DNA sample there'll be billions of fragments of DNA that have come from a patient, or from someone in the Biobank project. What we've done is attached short pieces of chemically-synthesised DNA to each end of those DNA fragments.

Phil - And it’s always the same two sequences?

Vince - It's always the same two sequences. We call them adaptor DNAs. They will then bind very specifically to a complementary piece of DNA on the surface of the flow cell, and that's the mechanism by which we attach DNA to the surface so that it's ready to sequence. You remember that DNA base pairs bind to each other, and we're making use of that property to attach the DNA very specifically to the flow cell surface.

Phil - And then what?

Vince - Then what we need to do is that we need to make copies of that one piece of DNA in each well. We end up with about a thousand pieces of DNA in each well. And having a thousand pieces of DNA in that well means that you get a lot more signal.

Phil - How do you get them to multiply?

Vince - So we use a special trick, it's a process called bridge amplification. The first piece of DNA does something called bridging, it bridges over, bending over to form this bridge on the surface, and binds to another complementary strand of DNA on the surface of the flow cell. We then make another copy. That process repeats, and these molecules of DNA continue to walk around the surface with this bridging process. At each step when they do that we make a copy of them, and very quickly you end up with, you know, it's an exponential process, with a thousand or so copies of that one DNA molecule, all with the same sequence.

Phil - So to clarify, what’s going on is that at high temperatures in the machine, DNA - normally a paired double-strand - separates into two single strands. When each strand then bends over to form a bridge on the flow cell, they apply an enzyme that uses the bridge shape to create that strand’s complementary pair. And the pair then stands back up, and because it’s still hot, they separate again. Both single strands bend over, and you keep going. The enzymes that they use are really cool because they need to work in high temperatures, so Illumina often gets them from little bacteria that live in the deep ocean around thermal vents. Using them, this process makes thousands of DNA copies in each of the billions of wells on the flow cell.

Vince - So the next step is that we have these little chemical building blocks of DNA that we've modified in the lab. And what we've done to them is two things. First of all we've added a fluorescent dye to them. The other thing we've done is added something called a terminator, or actually a reversible terminator.

Phil - That's not Arnold Schwarzenegger but he can do a three point turn?

Vince - Yeah, nothing to do with Arnie. It's a chemical modification to the natural building block of DNA that stops you adding more than one base at a time to a growing DNA strand. If we didn't have it there what would happen is the enzyme would just make a copy of the strand in its entirety in a single step. You wouldn't be able to see the DNA letter by letter.

Phil - What’s the enzyme?

Vince - So the enzyme is something called a DNA polymerase. In nature, you add a DNA polymerase to copy DNA and it will very rapidly just zip along the DNA strand and make a copy. In our system we at one building block of DNA at a time. We then image the whole flow cell to see which colour of base is lighting up. Then what we can do using another chemical process is remove the dye, the fluorescent dye, and also remove this terminator. And the process repeats…

Phil - Rinse, repeat.

Vince - Exactly.