Gel electrophoresis | Biomolecules | MCAT | Khan Academy

Let's say that you have some vials here, and you know that in the solution you have fragments of DNA in each of these. What you're curious about is, well, what about the DNA fragments in this first vial, in vial number one? How long are those fragments? How many base pairs long are they? You might say, "Well, why don't I just take them out and count them?" Except for the fact that they are incredibly small and incredibly hard to handle.

Even a fairly large fragment of DNA, let's say we're talking about something that's on the order of 5,000 base pairs, well that's going to be approximately 1 to 2 micrometers long if you were to completely stretch it out. We can't even start to think about how thin the actual diameter is. Lengthwise, the long way, it's only going to be 1 to 2 micrometers, which is super duper small. This is 1 to 2 thousandths of a millimeter, so that's not going to help us to somehow try to manipulate it physically with our hands or with rough tools.

So how do we do that? We could have other vials there. How do we see how long the DNA strands that are sitting in those vials actually are? The technique we're going to use is gel electrophoresis. It actually could be used for DNA strands; it could be used for RNA; it could also be used for proteins—any of these macromolecules—to see how long those fragments are.

And so let me write this down: gel electrophoresis. It's called gel electrophoresis because it involves a gel, and it involves electric charge. The "fesis" just refers to the fact that we're going to cause the DNA fragments to migrate through a gel because of the charge. So "fesis" is referring to the migration or the movement of the actual DNA.

How do we do this? Well, here is our setup. Right over here, we have our gel that's embedded in a buffer solution. This gel, the most typical one, is agarose gel. That's a polysaccharide that we get from seaweed, and it's literally a gel. It's a gelatinous material. What we're going to do is take samples, so we might take a little sample from this one right over here and we'll put it in this well right over here.

You can view these wells as little divots in the gel. You could take a little sample from here and put it into this well, and then you could put a sample from here and you could put it in that well. It's going to be bathed inside this buffer. You can see the buffer; I drew this fluid, and that's really just water with some salt in it. The buffer is going to keep the pH from going too far out of bounds as we place a charge across this entire setup.

If the pH gets too far in the basic or acidic side, it might actually affect the DNA or affect the charge on the DNA. What we're going to do is we're going to put a charge across this entire setup, where the side where the wells are, where we're going to place the DNA, that's going to be where we're going to put the negative electrode. So that's our negative electrode there, and the other end is going to be our positive electrode.

We're going to use the fact that DNA has a negative charge at the typical pH, right? Like the pH that we are going to be dealing with. We could go back into previous videos and we can see it right over here. You see these negative charges on our phosphate backbone.

So what is going to happen? What is going to happen once we connect both of these to a power source? This side is negative and this side is positive. Well, the DNA is going to want to migrate. Now let's think about what will happen. Will shorter things migrate further, or will longer things migrate further? You might say, "Well, longer things are going to have more negative charge, so maybe they go farther away." But then you also have to remember that they're also moving more mass, so their charge per mass is going to be the same regardless of length.

What determines how far something gets, how much it migrates over a certain amount of time, is how small it is. Remember, we have this agarose gel, and people are still studying the exact mechanism of how this DNA or these macromolecules actually migrate through the polysaccharide. If you imagine this polysaccharide is kind of a mesh, a net, a sieve, well, smaller things are going to be able to go through the gaps easier than the larger things.

So if you let some time pass, some of the DNA—let's say this DNA gets around there. I'm just color coding. You actually wouldn't see these colors; let's say this DNA gets around that far, so it doesn't get as far. Let's say that this DNA doesn't migrate; let's say it has some that migrates that far, and let's say it has some that migrates that far.

If you just saw this, you wait some amount of time, and you were to come back, and you were to see this migration occur. The longer you wait, the further these things are going to get. In fact, if you wait too long, they're going to fall off all the way over the other edge. If you just saw this, you'd say, "Okay, well, this strand right over here, these must be smaller DNA molecules. They must be shorter. These must be a little bit longer, and these must be even longer than that."

This grouping right over here is going to be the longest of all. This was a mixture of some longer strands and still longer ones, but not quite as long. For example, maybe there are some really short strands. Maybe there were some really short strands in that, what I'm drawing as that orange group right over here.

What I just did right over here—this could tell you the relative length of these strands. But how would you actually measure them? That's where you can go find standardized solutions, which we call a DNA ladder. So let's say you go get the DNA ladder. I'm going to draw it in pink. You literally could buy this; you could even buy it online.

The standard solution, let's say it separates like this. It separates like that, goes there; let's say some of it goes like there, and some of it goes like there. You would be able to know from the labeling, or whichever one you choose to buy, that this grouping here—that's all of the DNA that is 5,000 base pairs. Let's say this right over here is 500 base pairs, and let's say this over here is 500 base pairs long.

Now you can use this DNA ladder, these standardized ones, to gauge how long the DNA—how many base pairs these are. You say, "Okay, this blue one here; this is a bunch of DNA that's a little bit longer than 500 base pairs, but it's shorter than 1,500 base pairs." You can see this green one here, well, it's a little bit longer than 1,500 base pairs. It didn't migrate quite as far as this big bundle of 1,500 base pairs that did, and so then you can get a better approximation.

You can choose your ladder based on what you think you are going to find there, what you are actually going to look for. Now the other thing to appreciate is when you see the DNA having migrated this far, you might say, "Okay, is this one DNA strand? Is that one DNA strand that I'm looking at?"

Just going back to the measurements, no, that is many, many, many, many DNA strands that you're looking at. They're not all stretched out like that. Remember, even something that is 5,000 base pairs long is only going to be 1 to 2 micrometers if you stretch it out. You wouldn't be able to see that; it's a thousandth of a millimeter. You wouldn't even be able to see it, so this is many, many molecules of DNA migrating that far.

They would have to be that small to be able to migrate through that polysaccharide gel. Now the last thing you're probably saying is, "Okay, wait, but how am I even seeing it over here? How do I actually see this DNA, especially if they're these super, super small molecules?" The answer is, you put some type of marker on the DNA that will make them visible, some type of dye or something that might become fluorescent.

One of the typical things that people often use is ethidium bromide. Ethidium bromide is called an intercalating agent, and it's a molecule. You can see the ethidium right over here. These are two DNA backbones of DNA; you can see the base pairs bonding here, and then this right over here is ethidium that has fit itself—hence why we call it intercalating. It has fit itself in between the rungs of the ladder, and when it does so inside of DNA, it actually becomes fluorescent when you apply UV light to it.

So, if you put this ethidium bromide into all of your DNA right over here, and then as it migrates, and then if you were to turn on a UV light, it would become fluorescent, and you would actually see these things.

So if you wanted to see what it actually would look like in real life, well, this is what it would look like when you were to look at it straight on. Where this would have been a well—let me make it a little bit easier to read—so right over here would have been the well where you would put the DNA ladder, and it would come up with standardized measurements. Maybe that's our 5,000 base pairs, this right over here is our 1,500 base pairs, and this right over here is our 500 base pairs.

Then, let's say you had some solution of some other DNA, and you wait a little while, and you see, look, it migrated not quite as far as a 500 base pair, so it must be a little bit—this must be a bundle of things a little bit longer than 500 base pairs, but for sure a lot shorter than 1,500 base pairs.

Once again, it doesn't have to have just one fragment length. You could have had another group that was maybe right at 500 base pairs. You've probably seen this whenever you see people talking about genetic analysis and things like this—you're often seeing people look at one of these readouts from gel electrophoresis.

So now you know what's actually going on here. This isn't a strand of DNA; this is a big, a bunch of DNA that has been tagged with some type of dye, or the ethidium bromide or something like that, and it's a bunch of those molecules. They've migrated based on the charge. They're trying to get away from that negative charge to the positive charge. The smaller molecules—this is a bunch of small molecules right over here—are able to get further because they're able to get through the mesh of the agarose gel.