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Ligation of DNA
Quote
"Doctor Chromo doesn't know what he wants... one day he cuts DNA into pieces, the next, he puts it back together again."
Amoeba, September 97
Ligation described
Use of ligation in the recombination of DNA
Ligation described
There is nothing very difficult in the theory of ligation of DNA.
It is possible, using an enzyme called ligase
, to link pieces of DNA one after the other.
Any blunt end of a piece of DNA can be ligated to any other blunt end of DNA. So in theory, when a collection of fragments of DNA with blunt ends are put in the presence of ligase, after a while, one should find a solution with only one big piece of DNA. All the fragments would have ligated together.
If we could predict which fragments are going to ligate together, this could be really useful in molecular biology. One could take genes from different sources and ligate them together to create new genes, or modify them.
There is no way to predict what is going to link together, but there is a way to improve greatly the odds of two fragments linking together.
We have seen earlier that a restriction enzyme like EcoRI can create what are called sticky ends. When EcoRI cuts a DNA fragment, both strands of DNA are not cut at the same length, leaving four or more bases hanging single-stranded.
If one fragment of DNA has been cut by EcoRI, and is in presence of three kinds of DNA fragments (blunt end, overhung but not complementary, overhung and complementary (sticky)), ligation will be made between the ends that offer the most stable link. Figure 2 shows what happens.
On the left side of the image, there is a piece of DNA (blue) which has been digested with EcoRI. One of its ends has four bases hanging.
On the other side, there are other fragments.
- The first one (A) has blunt ends, and will not easily ligate to it.
- The next one (B) also has a four bases hanging, but they are not the complementary bases of the EcoRI digested fragment. These two fragments will have difficulty ligating.
- The last fragment (C), has four bases hanging, and they complement the four bases hanging of the EcoRI digested fragment. An hybridisation can happen between these two fragments. Although not very stable, it is more stable than the blunt/blunt pairing seen at the very beginning of this lesson. The ligase will stabilise the pairing, and make the join.
Use of ligation in the recombination of DNA
Recombinant DNA is made by linking together fragments of DNA from different sources. e.g. one of the fragments is a plasmid, which has been cut (linearised) and the other fragment can be any piece of DNA: A gene or an unknown portion of a genome. Figure 3 illustrates this definition, and also shows a theoretical way to make recombinant DNA
If we want to make some recombinant DNA, we need a solution containing both sorts of DNA (plasmid and insert), and ligase. The way the DNA has been fragmented / digested will affect the success of the ligation. A successful ligation should result in the production of a majority of the desired recombinant.
- To digest plasmids and inserts one can use:
- One enzyme that gives blunt ends
- One enzyme that gives sticky ends, (using the same enzyme for the insert and the plasmid)
- Two enzymes that give sticky end (different restriction site) using the same two enzymes for both the plasmid and the insert.
What will happen in these three cases when you come to ligate plasmid and insert.
- Case 1: Blunt ends In the introduction we saw that blunt ended DNA can ligate to any blunt ended DNA. If we use an enzyme that gives blunt ends to digest DNA and use the fragments straight away in a ligation, we will obtain a mixture of an infinite number of constructs, some of them could be quite big.
- Case 2: one enzyme / sticky ends. If we use one single enzyme to cut both the plasmid and the insert, we are left with almost the same situation as above: this is illustrated in figure 4. The most likely ligation that could happen is what is called self-ligation, when both ends of the same fragment of DNA, meet together and are ligated. (see figure 5)
- Case 3: double digest, sticky ends. If each fragment is cut by two enzymes which have different restriction sites, self ligation will not be possible, because the two ends of each fragment will not be complementary, or compatible. When the two fragments are put together in solution they will ligate together to form the desired recombinant DNA. It is possible to carefully plan a digestion/ ligation experiment in order to obtain the desired construct
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