Video Transcript
The diagram provided shows the
basic process of cloning a DNA sequence using a bacterial plasmid as a vector. What enzyme joins the
sugar–phosphate backbone of the DNA and the plasmid together in step 2?
This question provides a diagram
outlining the process used to clone DNA. In order to answer this question,
let’s review some basic terminology that relates to cloning DNA.
Cloning DNA often involves forming
recombinant DNA. Recombinant DNA is the combination
of DNA from at least two different sources. In the provided diagram, we can see
one source of DNA from a bacterium and the other from a human chromosome. The bacterial DNA, shown in orange,
is combined with human DNA, shown in green. This bacterial DNA is a special
type of DNA called plasmid DNA. Plasmids are extrachromosomal
pieces of bacterial DNA, meaning they’re separate from the bacterial chromosome. Plasmids can carry accessory genes,
like antibiotic resistance, that can help bacteria adapt to their environments. They replicate independently of the
bacterial chromosome.
These two sources of DNA are
combined to form the recombinant DNA that we see here. This recombinant DNA can be
transferred to bacterial cells where clones of it will be made as the cells divide
and the plasmid itself replicates. This is why we call this process
DNA cloning.
In order for recombinant DNA to be
made, the DNA from the two sources need to be cut. To cut DNA, we use special enzymes
called restriction enzymes. Restriction enzymes can recognize
and cut specific DNA sequences called recognition sequences. For example, the restriction enzyme
EcoRI cuts at the site GAATTC, which we can see in the black box here. When EcoRI cuts DNA, it cuts it in
the pattern shown here. When DNA is cut by a restriction
enzyme, it cuts the DNA by cleaving the phosphodiester bond in the sugar–phosphate
backbone of DNA, which we can see here. So this is how our plasmid and
human DNA can be cut, which we can see in our provided diagram.
Now let’s talk about how this DNA
can be joined together. After the sequence is cut, you’ll
notice that there’s overhangs, or regions of unpaired DNA bases. These are called sticky ends
because they have a tendency to stick back together due to the complementary base
pairs. So if we cut our bacterial plasmid
DNA and human DNA with the same restriction enzyme, we can bring them back together
because of the complementary sticky ends. In the diagram on the left, we can
see the sticky ends on the cut human DNA indicated here with pink arrows. And we can see the sticky ends on
the cut plasmid DNA here.
Because these ends are
complementary, since they were cut with the same restriction enzyme, they can be
joined together as we see here. When these two sticky ends come
together, there is still a gap in the sugar–phosphate backbone that was introduced
by the restriction enzyme. To repair this gap and bring the
sugar–phosphate backbone together, we need to use another enzyme called DNA
ligase. Once these sugar–phosphate
backbones are joined, the two sources of DNA are completely joined together and can
be transferred into bacterial cells for DNA cloning.
Going back to our question, the
enzyme that joins the sugar–phosphate backbone of the DNA and the plasmid together
in step 2 of the provided diagram is DNA ligase.
