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?