Restriction Enzymes and DNA Ligases Allow Insertion of DNA Fragments into Cloning Vectors
FIGURE 6-11 Cleavage of DNA by the restriction enzyme EcoRI. This restriction enzyme from E. coli makes staggered cuts at the specific 6-bp palindromic sequence shown, yielding fragments with single-stranded, complementary 4-base “sticky” ends. Many other restriction enzymes also produce fragments with sticky ends.
A major objective of DNA cloning is to obtain discrete, small regions of an organism’s DNA that constitute specific genes. In addition, only relatively small DNA molecules can be inserted into any of the available vectors. For these reasons, the very long DNA molecules that compose an organism’s genome must be cleaved into fragments that can be inserted into the vector DNA. Two types of enzymes—restriction enzymes and DNA ligases—facilitate production of such recombinant DNA molecules.
Cutting DNA Molecules into Small Fragments Restriction enzymes are endonucleases produced by bacteria that typically recognize specific 4–8-bp sequences, called restriction sites, and cleave both DNA strands at these sites. Restriction sites commonly are short palindromic sequences; that is, the restriction-site sequence is the same on each DNA strand when read in the 5′ to 3′ direction (Figure 6-11).
For each restriction enzyme, bacteria also produce a modification enzyme, which protects a host bacterium’s own DNA from cleavage by modifying the host DNA at or near each potential cleavage site. The modification enzyme adds a methyl group to one or two bases, usually within the restriction site. When a methyl group is present there, the restriction endonuclease is prevented from cutting the DNA. Together with the restriction endonuclease, the modification enzyme forms a restriction-modification system that protects the host DNA while it destroys incoming foreign DNA (e.g., bacteriophage DNA or DNA taken up during transformation) by cleaving it at all the available restriction sites.
Many restriction enzymes make staggered cuts in the two DNA strands at the corresponding restriction site, generating fragments that have a single-stranded “tail” at both ends (see Figure 6-11). The tails on the fragments generated at a given restriction site are complementary to those on all other fragments generated by the same restriction enzyme. At room temperature, these sticky ends can transiently base-pair with those on other DNA fragments generated with the same restriction enzyme.
The DNA isolated from an individual organism has a specific sequence that, purely by chance, contains a specific set of restriction sites. Thus a given restriction enzyme will cut the DNA from a particular source into a reproducible set of fragments called restriction fragments. The frequency with which a restriction enzyme cuts DNA, and thus the average size of the resulting restriction fragments, depends largely on the length of the recognition site. For example, a restriction enzyme that recognizes a 4-bp site will cleave DNA an average of once every 44, or 256, base pairs, whereas an enzyme that recognizes an 8-bp sequence will cleave DNA an average of once every 48 base pairs (65 kb). The hundreds of different restriction enzymes that have been identified from different species of bacteria allow DNA molecules to be cut at a large number of different sequences corresponding to the recognition sites of these enzymes (Table 6-1).
Inserting DNA Fragments into Vectors DNA fragments with either sticky ends or blunt ends can be inserted into vector DNA with the aid of DNA ligase. During normal DNA replication, DNA ligase catalyzes the end-to-end joining (ligation) of short fragments of DNA. For purposes of DNA cloning, purified DNA ligase is used to covalently join the ends of a restriction fragment and vector DNA that have complementary ends (Figure 6-12). The vector DNA and restriction fragment are covalently ligated together through the standard phosphodiester bonds of DNA. In addition to ligating complementary sticky ends, the DNA ligase from bacteriophage T4 can ligate any two blunt DNA ends. However, blunt-end ligation is inherently inefficient and requires a higher concentration of both DNA and DNA ligase than does ligation of sticky ends.
FIGURE 6-12 Ligation of restriction fragments with complementary sticky ends. In this example, vector DNA cut with EcoRI is mixed with a sample containing restriction fragments produced by cleaving genomic DNA with several different restriction enzymes. The short base sequences composing the sticky ends of each fragment type are shown. The sticky end on the cut vector DNA (a′) base-pairs only with the complementary sticky ends on the EcoRI fragment (a) in the genomic sample. The adjacent 3′ hydroxyl and 5′ phosphate groups (red) on the base-paired fragments are then covalently joined (ligated) by T4 DNA ligase.