Video Transcript
In this video, we’ll discuss
cloning of DNA sequences. First, we’ll define DNA cloning and
recombinant DNA and then look at how plasmids can be used together with restriction
enzymes and DNA ligase during the cloning process. Then, we’ll look at how bacterial
cells can be transformed with recombinant DNA to make the clones. Finally, we’ll examine how reverse
transcriptase and PCR can be used to isolate genes of interest for cloning.
The nucleated cells in our body
contain DNA that give us the instructions needed for life. These instructions are organized as
genes in DNA that control the expression of many of our traits. They can control the color of our
eyes, our height, and of course our health and whether or not we’re affected by a
disease. For example, the gene for insulin
can produce a hormone that can control blood glucose levels.
Insulin is able to signal to the
cell to take up glucose so it can be used for energy. A disease called diabetes can limit
the amount of insulin in the body. And without sufficient insulin, the
cell isn’t able to take up the glucose that it needs to carry out its functions. So, for diabetics, insulin needs to
be given periodically to ensure that cells take up the glucose that they need. So where do we get this insulin
from?
We can extract it from animals,
like pigs, believe it or not. And this was common for many years
because pig insulin is similar to human insulin. However, allergic reactions were
still possible. So pig insulin was not ideal. We found our pig insulin
replacement in the 1970s using something you probably wouldn’t expect, bacteria. So how is this possible? Well, we can take our gene for
human insulin and insert it into bacterial DNA. The bacterium will then go on to
produce human insulin. This can then be extracted and
purified and used to treat diabetes.
Now is a good time to define a
couple of key terms we’ll be using in this video. So let’s clear some room on the
left of the screen. This process that we just described
is referred to as DNA cloning. Here, the human insulin gene is
inserted into bacterial DNA. And this is actually creating a
genetically identical copy or a clone of the insulin gene. We can also use this as a verb and
say that we cloned the insulin gene in this example.
Our second definition is
recombinant DNA. When DNA cloning, we often use
bacterial DNA to carry our gene of interest, or insulin in this example. So we have DNA from two sources:
one from human DNA, as shown here in green, and one from bacterial DNA, shown here
in black. These two sources of DNA are then
combined together to form what we call recombinant DNA.
Now that we’ve covered DNA cloning
in a general way, let’s talk more about the details of this process. We’ll start with discussing how
recombinant DNA can be made. So here’s our recombinant DNA
that’s been combined from two sources. We could see our gene of interest
in green and the bacterial DNA in black. This bacterial DNA comes from a
special type of DNA called plasmid DNA. If we look inside a bacterial cell,
we’ll notice that there’s two types of DNA. There’s the bacterial chromosome,
which is often a large circular piece of DNA that contains the information needed
for the life cycle of the bacterium. And there’s plasmid DNA, which are
also circular in structure and can act as accessory DNA molecules that can be shared
between bacteria.
Plasmids may contain certain genes,
such as antibiotic resistance genes, that can be beneficial for bacteria to grow in
certain conditions and can also be used as a method that scientists use to select
for bacteria that take up the plasmid. These plasmids are able to
replicate independently of the chromosomal DNA. And a single bacterium can have
hundreds of copies of a particular plasmid.
Plasmid DNA can also be called
carrier or vector DNA because it can carry our gene of interest and can be taken up
by our bacterium, where it’s treated like an ordinary plasmid. This means that it can express our
gene of interest and also make multiple copies or clones of this gene as the plasmid
replicates. In addition, these plasmids are
passed on as bacteria divide, making even more copies.
Now, let’s talk about how plasmid
DNA and our gene of interest can be joined to make recombinant DNA. So we’ll start with our plasmid
DNA. But remember, DNA is double
stranded. So let’s draw this to be a little
bit more accurate. Okay, that looks better. And here we have our second source
of DNA containing our gene of interest. So, in order to get our gene of
interest inside the bacterial plasmid, we’re gonna need something to cut the DNA so
we can insert this gene. And what cuts better than a pair of
scissors? Molecular scissors, that is. You’ll recall that restriction
enzymes or restriction endonucleases are special enzymes that act as molecular
scissors to cut DNA. So our plasmid DNA can be cut here,
and our gene of interest can be cut out from both sides.
Let’s see how this looks in the
actual sequence of DNA. We’ll start with the plasmid
DNA. Restriction enzymes cut DNA at
their recognition sequence. The sequence GGATCC is the
recognition sequence for the restriction enzyme BamHI. BamHI cuts DNA as indicated and
produces these two ends. These are called sticky ends
because they have unpaired nucleotides that can base-pair with complementary
nucleotides on an opposing strand of DNA. So the sequence GATC is
complementary to the sequence CTAG. This means that these two ends can
hydrogen-bond with each other, which is what makes them sticky.
Now that we’ve cut our DNA on the
right, let’s see what this looks like in the plasmid DNA diagram on the left. There, you’ll still notice that
this diagram here is also showing the two sticky ends. So this section here corresponds to
this sticky end, and this section here corresponds to this sticky end.
So now that we’ve opened up our
plasmid using the BamHI restriction enzyme, let’s do the same thing for our gene of
interest on the bottom. So now we can see the sequence on
the right with our gene of interest being bordered by the two BamHI recognition
sequences. And now we can cut these sequences
with our restriction enzyme BamHI to give the indicated cutting patterns. This releases our gene of interest,
which now has two sticky ends. These sticky ends are compatible
with the two sticky ends in the cut plasmid DNA. So now we can combine them. That looks better.
Now, let’s do the same thing to the
diagrams on the left to see how this looks. So the restriction enzyme cuts as
indicated here, which can then be inserted into our cut plasmid DNA, as you can see
here. So you may have noticed that
there’s gaps in the diagram on the left, one of which is indicated by this pink
square. We can also see this in the
sequence on the right. These gaps represent missing
phosphodiester bonds in the sugar phosphate backbone of DNA.
Let’s zoom in and see what this
means. Here, we can see the chemical
structure of the two strands of DNA. The strand on the left is the five
prime to three prime strand. And on top, this corresponds to the
top sequence, whereas the bottom sequence is the three prime to five prime strand,
which corresponds to the structure on the right in the bottom diagram.
Nucleotides are joined together by
phosphodiester bonds as indicated here. Restriction enzymes cleave this
phosphodiester bond, and this is where this gap comes from. So, at this point, the only thing
holding our gene of interest in the plasmid is the hydrogen bonding between the
sticky ends. To combine these two DNA molecules
permanently, we need to repair this missing phosphodiester bond. To do this, we can use an enzyme
called DNA ligase, which can catalyze the formation of a phosphodiester bond. DNA ligase can therefore fill in
all the gaps in the sugar phosphate backbone that were introduced by the restriction
enzyme. You can see this here on the top
and here on the left as well.
Now, our recombinant DNA is all
stitched up and ready for the next step, where we transfer this into bacteria. This step is known as
transformation. During transformation, the bacteria
are exposed to a certain chemical and temperature that can help make them more
permeable to DNA. This way, when the bacterium is
mixed with the recombinant DNA, it is readily taken up into the cytoplasm.
Now, the recombinant DNA can be
expressed using bacterial components to form the corresponding protein from our gene
of interest. If the gene was insulin, for
example, we’d now be able to harvest the insulin protein. A single recombinant DNA molecule
is shown here for simplicity. But there will actually be multiple
copies, since it will replicate just like a plasmid would. In addition, the bacteria will
divide to make even more copies. Ultimately, this will produce a
large amount of our protein.
Now that we’ve seen how DNA cloning
takes place, the next topic is how to isolate our gene of interest in order to clone
it. Normally, genetic information flows
from DNA to mRNA to protein. A gene in DNA can be converted to
mRNA by transcription. The reverse process can also take
place, where mRNA is converted back into DNA. This process is called reverse
transcription. So, if we want to clone a
particular gene, one way to do this is to isolate the mRNA and reverse-transcribe it
back into DNA.
It’s important to be working with
DNA because restriction enzymes and plasmids, which we need for cloning, are usually
exclusively DNA based. So how do we isolate this mRNA in
order to make the DNA for our gene of interest?
Suppose our gene of interest is
insulin. We know that the pancreas contains
some cells that produce a lot of insulin mRNA. So this is a good place to
start. In the lab, we could harvest these
cells and isolate this mRNA. We can then perform reverse
transcription on the mRNA to make insulin DNA that we can then insert into a plasmid
for DNA cloning.
Now, let’s write up the sequence of
mRNA so we can work out how reverse transcription actually takes place. So here is our mRNA, and here is
the enzyme that performs reverse transcription that we call reverse
transcriptase. Because it adds DNA nucleotides
that are complementary to mRNA, we call its product cDNA or complementary DNA. As reverse transcriptase moves
along the mRNA in the five prime to three prime direction, it adds these
complementary nucleotides to the growing cDNA molecule.
Now that the cDNA molecule is
complete, let’s take a second to go over a few things. Remember that mRNA contains uracil
in place of thiamine as in DNA. And uracil base-pairs with adenine
as shown here. In DNA, we have thiamine, which can
base-pair with adenine in mRNA. A common mistake is to think that
this adenine would pair with uracil, but reverse transcription produces cDNA and not
mRNA. So we want to be sure that we’re
using the right nucleotides.
Now, what we want to do is get rid
of the mRNA in this mRNA and cDNA complex and replace it with another strand of DNA
to make this whole molecule double-stranded DNA. That way, we can use it for DNA
cloning. So, in the lab, we can use a
special enzyme to specifically degrade this RNA molecule to remove it.
Now, with mRNA gone, we’re left
with the single-stranded cDNA. The next step is to make this DNA
double stranded. And for that, we can use our old
friend DNA polymerase, who you might remember plays a big part in replicating DNA
before cell division. So DNA polymerase can bind to the
single-stranded cDNA molecule and add complementary nucleotides to make the second
strand of DNA. And now we have our double-stranded
DNA molecule that’s ready for DNA cloning.
We can also make multiple copies of
a DNA molecule using another technique called the polymerase chain reaction, or
PCR. This is a lab technique that allows
us to target specific regions of an organism’s DNA to make multiple copies of
it. By doing this, we’re actually able
to make clones of our gene of interest without even using bacteria. However, in some situations, we
might want to use these copies to form recombinant DNA, which can then be used to
transform bacteria. This is useful because the protein
can then be made. So, in this way, PCR is another way
that we can isolate our gene of interest.
Now that we’ve seen how cloning
works and how to isolate genes to be used in cloning, it’s time to look at a
practice question.
Using plasmids to form recombinant
DNA is a crucial part of cloning DNA. In which microorganisms were
plasmids originally discovered?
Let’s look at an example of cloning
the interferon gene in order to answer this question. Interferons are proteins that can
interfere with viral replication. As such, they can be used
effectively as antiviral drugs. They can also be used to treat
certain diseases, such as cancer and multiple sclerosis. In the past, interferon was
extracted from cells, which made it very expensive. These days, a process called DNA
cloning can be used. This makes it possible to make a
copy or clone of the gene for interferon. This process starts with isolating
the gene for interferon and inserting it into a circular piece of bacterial DNA
called a plasmid.
Plasmids were originally discovered
in bacteria and are extrachromosomal pieces of DNA that replicate independently. A single cell can have hundreds of
copies of a single plasmid. In cloning, they’re used to carry
genes that we’re interested in making copies of. So, in this context, they’re
sometimes called vectors or carrier DNA. On the left, you’ll notice that
this construct we’ve made contains DNA from two sources. One is the DNA from the human
interferon gene, and the other is the plasmid DNA from the bacteria. When two sources of DNA are
combined like this, we call it recombinant DNA.
In the final step of the cloning
process, this recombinant DNA can then be transferred into bacterial cells. Here, the bacterial cell can
express this gene to produce the interferon protein. Only one copy of the recombinant
DNA is shown here. But it will replicate in the cell
to make many copies. And there will be even more copies
as the bacteria divide. This will lead to a lot of
interferon production, which can then be extracted and used for medical
purposes. Plasmids are a crucial part of DNA
cloning because they’re used to carry the gene we want to clone. Plasmids were originally discovered
in bacteria.
Now, let’s go over some of the key
points that we covered in this video. DNA cloning is the process of
making copies or clones of specific DNA that we’re interested in. This DNA can be cloned into a
plasmid. These two sources of DNA, the
specific DNA we’re interested in and the plasmid DNA, can be combined to form
recombinant DNA. Recombinant DNA can be made by
cutting DNA using restriction enzymes and then inserting this in the plasmid
DNA. DNA ligase can then be used to form
new phosphodiester bonds in the cuts made by the restriction enzyme. Recombinant DNA can then be
transformed into bacteria, where multiple copies can be made and expressed using
bacterial components. Finally, the specific DNA that
we’re interested in cloning can be isolated using reverse transcription or PCR.