Figure 2.6. The mechanism of DNA joining by DNA ligase. See the text for details. This figure is adapted from Doherty et al. (1996)
2.3 The Basics of Cloning
The ability to break and rejoin DNA molecules almost at will led to the first experiments in DNA cloning in 1972 (Jackson, Symons and Berg, 1972).
Figure 2.7. Breaking and joining DNA using restriction enzymes and DNA ligase. Linear DNA (insert) and a closed-circular plasmid DNA (vector) each contain the recognition site for BamHI and EcoRI. Mixing the DNA fragments with compatible ends together in the presence of DNA ligase can result in the formation of vector-insert hybrid DNA molecules
For the first time it was possible to extract a fragment of DNA from one source and insert, or clone, it into the DNA from another source. Perhaps the most common type of cloning experiment involves the insertion of a foreign piece of DNA into a suitable vector so that the foreign DNA may be propagated in E. coli. In Chapter 3 we will discuss the various different types of vector that are available, but at this stage we could consider the vector as a closed-circular double-stranded plasmid DNA molecule. If we wish to insert foreign DNA sequences into this vector, we need to cut it to produce a linear DNA onto which we can attach other DNA sequences using DNA ligase.
Let us first consider the insertion of DNA into the vector using two different restriction enzymes (Figure 2.7). Treatment of both a vector and insert DNA sequences with the restriction enzymes will generate a number of DNA fragments. In the vector the recognition sites for the restriction enzymes are located close to each other. As we will see in Chapter 3, this is very common in engineered plasmids. Cutting such a vector with BamHI and EcoRI will yield two fragments - a large one, comprising the majority of the vector, and a small one, representing the DNA between the restriction enzyme recognition sites. In the presence of DNA ligase, neither of these fragments is able to ligate to itself because the DNA ends are not compatible with each other. Digestion of the linear insert DNA sequence with BamHI and EcoRI results in the generation of three DNA fragments. Only one of the fragments contains a BamHI- and EcoRI-compatible end; the others represent the DNA at either end of the fragment. Mixing the vector DNA and insert DNA that are compatible with each other will result in the formation of hydrogen bonds between the two DNA molecules. If the ends were not compatible, this hydrogen bonding would not occur. The addition of DNA ligase to the hydrogen bonded intermediate will result in the sealing of the DNA backbone and the formation of a vector-insert hybrid DNA molecule. If one of the other insert DNA fragments becomes hydrogen bonded to the vector, say via its BamHI-compatible end, then ligation will not result in the formation of a closed-circular vector. As we will see in the next section, such DNA molecules are not replicated when they are transformed into bacteria.
This type of cloning scheme works because the vector DNA that has been cut with the two restriction enzymes contains non-complementary DNA ends. If the vector had been cut using a single restriction enzyme, or with two restriction enzymes that left the same sticky ends, then the vector could easily recircularize in the presence of DNA ligase. Treating the vector with a phosphatase enzyme after it has been cut with the restriction enzymes, however, can prevent this. Phosphatases catalyse the removal of 5' phosphate groups from nucleic acids and nucleotide triphosphates. Since phosphatase treated DNA fragments lack the 5' phosphate required by DNA ligase, such treatment will inhibit vector self-ligation and will promote the formation of vector-insert DNA hybrids. Such a cloning scheme is shown diagrammatically in Figure 2.8. When the vector has been treated with phosphatase, DNA ligase
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