ABSTRACT
After DNA has been extracted and quantied, it can be manipulated and studied in several ways. One of the initial characterizations that can be performed is analysis using restriction enzymes. The enzymes are specic to each bacterial species, have specic sequences that they recognize and cut, and have evolved to digest primarily foreign DNA. The bacterium that produces the enzyme protects its own DNA by adding protecting groups onto the recognition sequences. The enzyme will then digest the foreign DNA, but the DNA from the organism itself will be protected by the protecting group on the DNA. The protecting group is usually a methyl attached to one or more of the bases. The names of the enzymes are usually based on the bacterial species from which they were isolated. For example, EcoRI was the rst restriction endonuclease isolated from Escherichia coli. Some make staggered cuts that leave the nucleotides on the 5′ ends exposed (e.g., EcoRI, BglI, and BamHI; Figure 20.1), some leave the 3′ nucleotides exposed (e.g., HhaI, KpnI, and PstI), and some make blunt-end cuts (e.g., AluI, EcoRV, and HaeIII). When one or more of the bases are methylated, many will fail to cut at those spots. However, a few cut only when one or more bases are methylated. Most require magnesium ions, specic salt concentration ranges, physiological pH ranges, and narrow temperature ranges to function properly. Many can be stopped by adding EDTA (to chelate the magnesium) and/or by heating to more than 65°C. Most are inhibited by certain polysaccharides (found in plants and fungi), as well as by some other molecules. If the proper environment is not provided for the enzymes, they may cut inefciently causing partial digestion of the DNA, or they may completely fail to cut the DNA. For some, altered conditions may cause the enzyme to recognize and cut slightly different sequences (often called star or asterisk activity). Because the enzymes cut at specic sites and much of the DNA changes little, these enzymes can cut the DNA into fragments of reproducible lengths. When separated on agarose gels and hybridized to labeled gene probes, maps of the DNA regions of interest can be produced, based on their restriction enzyme sites.
