How Ligase is Used to Engineer Recombinant DNA

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  • 0:03 Genetic Engineering
  • 0:50 Review of DNA Ligase…
  • 1:45 DNA Ligase in Genetic…
  • 2:54 Mechanism of DNA Ligase
  • 4:58 Lesson Summary
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Lesson Transcript
Instructor: Greg Chin
DNA ligase makes recombinant DNA technology possible. In this lesson, you will learn how new versions of genes can be designed for experiments in novel host organisms using DNA ligase.

Genetic Engineering

The goal of genetic engineering is changing the genetic makeup of an organism. Recall that we previously learned that a restriction enzyme is an enzyme that can cut DNA and that these enzymes are commonly used in genetic engineering experiments. Scientists can use restriction enzymes to excise a specific gene from a genome and add it to a DNA plasmid to create a completely new recombinant version of the gene.

But, restriction enzymes just provide half of the story, right? To actually create recombinant DNA, pieces of DNA must be pasted together. That's where the enzyme DNA ligase comes in.

Review of DNA Ligase in Replication

If you recall, nature encountered a similar challenge to the one we're facing as scientists. During DNA replication, a cell makes an exact copy of each DNA molecule. However, because DNA polymerase (the enzyme that builds new DNA molecules) can only function in one direction with respect to DNA, a cell must treat each side of the replication fork slightly differently. At the leading strand, DNA polymerase can add nucleotides to build the new DNA molecule without stopping.

In contrast, as the replication fork opens, discontinuous fragments of DNA are built at the lagging strand. These fragments are then glued, or ligated together, by DNA ligase.

Discontinuous fragments on the lagging strand are bonded together by DNA ligase.
Ligase

DNA Ligase in Genetic Engineering

If restriction enzymes are like molecular scissors, then DNA ligase is like molecular glue. Scientists can use restriction enzymes to generate DNA fragments and DNA ligase to glue those fragments together. Let's see how this new experimental tool can be used in our human insulin experiment.

We can use restriction enzymes to cut the human insulin gene from a sample of human DNA. Then, we can cut a DNA plasmid with the same restriction enzymes.

Why is it important to use the same enzymes? Recall that the restriction enzymes we've discussed leave sticky ends when they're done cutting. Each sticky end will only base pair with a complementary DNA sequence. That means that, generally speaking, the sticky ends of different enzymes will be incompatible. Once hydrogen bonds between the complementary base pairs stabilize the structure, DNA ligase can glue the fragments together to form a single molecule.

We use the same restriction enzymes because the sticky ends will only bond with complementary bases.
Sticky Ends

Mechanism of DNA Ligase

Now, you may wonder why DNA ligase is even necessary. I mean, the hydrogen bonds are holding the two molecules together. That should be good enough, right? Recall, though, that hydrogen bonds are constantly broken and reformed during such cellular processes as replication and transcription.

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