Hi there I'm Jayme Dyer. This video is
about gel electrophoresis, which is a technique used to visualize large
quantities of DNA that have been separated by size. Gel electrophoresis is
used in a variety of research applications. Here is one example. In 2008
the very first person ever was cured of HIV. Cured of HIV! It turns out he didn't
just have HIV. He had leukemia too – which is a cancer of the blood. And the way that his doctors decided to treat him was to give him a bone-marrow transplant.
Essentially, they replaced his bone marrow with the bone marrow of a donor. Now, that helped cure the leukemia because the donor didn't have cancer of
the blood. But the way that it cured his HIV is that the donor was actually
genetically resistant to HIV. We all have a gene called CCR5. And about 1% of the
population has a variant of the gene that has a 32 base pair deletion. If both
of your copies of CCR5 have that deletion, then you are resistant to HIV.
But if you have just one of the longer variants you are susceptible to
infection by HIV. To try to cure the patient's HIV, the doctors used a bone-marrow donor who had two copies of the short, HIV resistant CCR5 variant. To
test whether they had successfully replaced all of his bone marrow with the
HIV resistant bone marrow, the scientists used PCR and gel electrophoresis to
quickly and easily visualize the size of his bone marrow CCR5 gene. So the researchers used PCR to amplify a portion of the patient's CCR5 gene. And they did this with a sample of his blood from before and after the bone marrow
transplant. And at the end of the PCR reaction, what they had was two PCR tubes one of which had lots of copies of his CCR5 gene from before the bone
marrow transplant and the other one had lots of copies of his CCR5 gene from
after the bone marrow transplant. And all they had to do was look in this tube after the bone marrow transplant to ask, "Are these pieces of DNA long or short?" And if they're long, then that would mean that he got a long CCR5 variant that is
sensitive to HIV. And if the pieces of DNA were short then that would mean he
got the short CCR5 variant that is resistant to HIV. So the researchers need
a way to see whether the pieces of DNA in here are long or
short. But the problem is that DNA is tiny. This little tube has one trillion
pieces of DNA in it. I can't see them by eye! I can't even see them with a light
microscope! So I need a way to see whether the pieces of DNA in this tube are long or short. And the cheap and easy way to do that is gel electrophoresis. It
works essentially like this: Imagine that this is a PCR tube full of pieces of DNA. And in this tube, I have two lengths of these pieces of DNA, I have short ones and long ones. So what I need to do is I need to tell whether all the pieces in
this are long or short or both. And so I'm going to put the pieces of DNA on a
mesh so I'm gonna use a hardware cloth in this case. So I'm going to pour my yarn, my pieces of DNA, on the mesh and then I'm gonna let gravity, I'm gonna do a little shaking. I'm gonna let gravity work and you'll notice that the pieces of DNA. The pieces of DNA, my yarn that falls through the mesh, are all short
pieces. Some of the long pieces start working their way through, but it's gonna
take some time before they can make it all the way through. Whereas the little
ones can sneak through the holes really easily. Now, gel electrophoresis is actually more like multiple layers of hardware cloth so that the little ones sneak their way through and the long
ones it takes longer for them to make it all the way through all the layers of
the mesh. Of course DNA is tiny you can't use hardware cloth to separate pieces
of DNA. You need something a lot smaller so what we use is a gel. That's where the
gel from gel electrophoresis comes from. This is a gel and you make it from this
powder. It's just a white powder. It's a carbohydrate that's isolated from
an algae. You mix it with water, heat it up in the microwave, let it cool. It's
basically like jello okay, but unlike jello this gel has holes that
are just the right size for DNA molecules to snake through. The problem
is, unlike my yarn example, we can't use gravity to pull the DNA through the gel.
We use, instead, an electric current. That's the electrophoresis part of gel
electrophoresis. An electric current pulls the DNA through the gel because
DNA has this big negatively charged phosphate on it which makes it attracted
to the positively charged pole in an electric current. To use the electric
current, I put the gel in this gel rig which is
full of buffer and then I apply electricity. And the electricity comes in
from the negative pole here. And it runs through the buffer through the gel to
the positive pole. And the DNA is negatively charged, so it's going to run
from up here to down here in the gel and that's going to help me separate the DNA
by size. Now I need to get the DNA in this gel somehow. And to do that I'll put
the DNA in this well, which is a little hole that I made when I made the gel. Here I am loading the DNA into the gel. I've added loading dye to the DNA
samples which lets me see the liquid as I pipette it into the gel.
Once I've loaded all my DNA samples, I turn on the current which pulls the DNA
through the gel. Remember that the DNA molecules are moving through a molecular mesh. The smaller pieces of DNA snaked through the holes of the mesh easier
than the bigger pieces. So the bigger pieces end up higher in the gel than the
smaller pieces. After about 30 to 45 minutes of running the electrical
current we can look to see where the pieces of DNA are. Now the purple stuff
there, that's the loading dye. That's not the DNA. To see the DNA, we use a stain
that binds to DNA and fluoresces under UV light. And that's exactly what the
researchers did with the PCR samples from the patient. I can't show you the
actual picture of the gel for copyright reasons, but here's another gel as an
example. The wells are at the top and the DNA ran from the top to the bottom of
the gel. This bright white band is those one trillion pieces of DNA made during
the PCR reaction which are bound to the fluorescent dye. Brighter bands, like this
one, have more DNA molecules in the band than less bright bands. The smaller DNA
pieces, like this one, moved faster so they're closer to the bottom of the gel
than the bigger pieces. This over here is the ladder which has known sizes of DNA
and is used like a ruler in a mug shot to tell how big the other pieces of DNA
are. When the scientists looked at the gel from the patient samples before and
after the bone-marrow transplant, they saw that before the bone-marrow
transplant he had a copy of the long, HIV sensitive CCR5 gene and after the
bone-marrow transplant he only had the short HIV resistant copy of CCR5. So
that's how the scientists knew that the bone marrow transplant had made him
resistant to HIV. So to recap, gel electrophoresis is a way
to see large quantities of DNA that have been
separated by size. DNA is loaded into the gel and an electric current pulls the
DNA from the top to the bottom of the gel. You can tell how much DNA is present
by how bright the band is. Brighter bands mean more DNA. And you can tell about how big the pieces of DNA are by where they are located in the gel. Bands that are
closer to the top of the gel have bigger pieces of DNA and bands that are closer
to the bottom of the gel have smaller pieces of DNA. You can tell about how big
the pieces of DNA are by comparing the height of the band in the gel to the
height of the bands in the DNA ladder. By the way, the patient has been free of HIV
and leukemia for over ten years and in 2019 another patient was cured of HIV in
the same way. What problems might you solve using gel electrophoresis?
