Question Video: Describing the Role of DNA Ligase in Forming Recombinant DNA | Nagwa Question Video: Describing the Role of DNA Ligase in Forming Recombinant DNA | Nagwa

Question Video: Describing the Role of DNA Ligase in Forming Recombinant DNA Biology • Third Year of Secondary School

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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?

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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.

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