This is Bob. Bob dreams of being a
world-renowned soccer player. He wants to play in the biggest leagues and in the
biggest games. But there’s a major issue stopping him from attaining this goal and
that’s his rare and severe case of hemophilia. He bleeds a lot from
everywhere. His skin is very sensitive and so he gets wounds often. He
experiences pain and swelling in his joints, mostly in his knees, elbows and
ankles. And when he goes to the bathroom there’s more blood to lose. So let’s get
into exactly what causes these symptoms. This is a DNA molecule and its genes
are responsible for giving us our characteristics. There are genes that are
responsible for making proteins called clotting factors. The clotting factors are
what help us clot blood and stop wounds from bleeding.
Unfortunately, for Bob, the gene for a certain type of clotting factor called
factor 7 is not working properly and that’s why he experiences these symptoms.
Now, let’s see the effect of this within the body. Here, we have a blood vessel and
of course it has plasma and red blood cells inside. The blood is flowing
normally, but let’s say you get a wound. There’s going to be a tear in the blood
vessel, making it lose a lot of blood. Now, what usually happens is the clotting
factors will stop the blood, helping in the sealing of the tear. But for Bob,
because they’re not working properly, and they’re not as abundant, they will not be
able to stop the bleeding. But how do we fix all of this. The answer is
recombinant DNA technology which refers to series of procedures used to join
together DNA segments from different species. And you’ll see what two DNA
segments we combine later on. In treating Bob’s case of hemophilia, we’re going to
use recombinant DNA technology to produce a bunch of clotting factors. And
this is done by taking the factor 7 gene and having it replicate inside bacteria.
We do this using a plasmid vector. A plasmid is a circular DNA molecule that
exists outside the bacteria’s main DNA. It can safely carry the clotting factor
gene into the bacteria and have it replicate. The plasmid also contains
genes that give the bacteria special advantages and one of these advantages
is antibiotic resistance. This plasmid in particular is a pBR322 plasmid and
has a tetracycline resistant gene and an ampicillin resistant gene. So the first
step for getting the factor 7 gene to replicate is to insert it into
the plasmid. This is done using restriction enzymes. They cleave in the
plasmid at locations called restriction sites which contain specific DNA
sequences recognized by the restriction enzymes. Each restriction enzyme
recognizes a specific sequence so when it comes across it, it will cut it from the
plasmid. In this case we’re using restriction enzyme eco r1.
Now that the eco r1 restriction enzyme has cut at its restriction site, we can
insert the factor 7 gene in the plasmid. This is now a recombinant DNA molecule
since we have combined DNA segments from two sources: the plasmid from the bacteria
and the clotting factor gene. The second step is to get the plasmid into the
bacteria which is done by bathing the plasmids and the bacteria in a calcium
chloride solution which makes it easier for the bacteria to take up the plasmids.
Let’s take a closer look at what exactly happens in the solution. As you can see
the bacteria and the plasmids are submerged and our goal is to get the
plasmid into the bacteria. Most of the bacteria actually won’t take up the
plasmid. It might be that only one or two take up the plasmid so that is why we put
in many copies. And even worse we can’t actually see which bacteria took up the
plasmid and which didn’t. In order to separate the ones that did take up the
plasmids and the ones that didn’t, we have to culture them in antibiotics. Why? Well,
remember the plasmid can give special advantages such as antibiotic resistance.
The plasmid we use had resistance to the antibiotic ampicillin. Only the bacteria
that took up the plasmid containing the ampicillin resistant gene would survive.
Slowly, each bacterium without the plasmid would
die. Once we have the bacterium with the plasmid, we can replicate it and those
bacteria can make the clotting factors since they all have the factor 7 gene in
their plasmid. After they make the clotting factors, we extract them and mix
them with sterile water so they can be injected. Now, we can give the injection
to Bob and he’ll feel much better. Maybe, just maybe, he could chase his soccer
dreams after all.