"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
There is nothing very difficult in the theory of ligation of DNA.
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.
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.
What will happen in these three cases when you come to ligate plasmid and insert.