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8.3: Cutting and Pasting DNA: Restriction Digests and DNA Ligation

8.3.1 Restriction Enzymes

Many bacteria have enzymes that recognize specific DNA sequences (usually 4 or 6 nucleotides) and then cut the double stranded DNA helix at this sequence (Figure 8.7). These enzymes are called site-specific restriction endonucleases, or more simply “restriction enzymes”, and they naturally function as part of bacterial defenses against viruses and other sources of foreign DNA. To cut DNA at known locations, researchers use restriction enzymes that have been purified from various bacterial species, and which can be purchased from various commercial sources. These enzymes are usually named after the bacterium from which they were first isolated. For example, EcoRI and EcoRV are both enzymes from E. coli. EcoRI cuts double stranded DNA at the sequence GAATTC, but note that this enzyme, like many others, does not cut in exactly the middle of the restriction sequence (Figure 8.8). The ends of a molecule cut by EcoRI have an overhanging region of single stranded DNA, and so are sometimes called sticky-ends. On the other hand, EcoRV is an example of an enzyme that cuts both strands in exactly the middle of its recognition sequence, producing what are called blunt-ends, which lack overhangs.

Figure 8.7:
An EcoRI dimer (blue, purple) sits like a saddle on a double helix of DNA (one strand is green, one is brown).  This image is looking down the center of the helix.

Figure 8.8:
The recognition sequence for EcoRI (blue) is cleaved by the enzyme (grey).  This particular enzyme cuts DNA at a position offset from the center of the restriction site.  This creates an overhanging, sticky-end.

8.3.2 DNA Ligation

The process of DNA ligation occurs when DNA strands are covalently joined, end-to-end through the action of an enzyme called DNA ligase. Sticky-ended molecules with complementary overhanging sequences are said to have compatible ends, which facilitate their joining to form recombinant DNA.  Likewise, two blunt-ended sequences are also considered compatible to join together, although they do not ligate together as efficiently as sticky-ends. Note: sticky-ended molecules with non-complementary sequences will not ligate together with DNA ligase. Ligation is therefore central to the production of recombinant DNA, including the insertion of a double stranded DNA fragment into a plasmid vector.