Recognition sequences for restriction enzymes are like signposts on a DNA molecule. Just as a highway map gives the important features along a road, restriction enzymes can give a map of features along a DNA molecule. Thus, for example, the circular DNA molecule of a small virus can be completely digested by a restriction enzyme to make a so‐called limit digest. Putting the fragments into a gel and passing electrical current through the gel separates the fragments. Because DNA has negatively charged phosphates at each phosphodiester bond, the positive direction of the gel will attract the DNA. The DNA fragments start to move, but the larger the fragment, the more often it “bumps into” the gel. As a result, the DNA fragments are separated by size, with the smallest fragments moving farther in the gel, as shown in Figure 1.
After the sizes of the fragments are known, they can often be ordered by several strategies. The simplest (in theory if not in practice) is to determine the size and composition of partial digestion products that contain two or more of the limit fragments. For example, if a DNA contains EcoRI fragments of 1500, 2000, and 3000 base pairs, a partial digestion that gives fragments of 3500 and 4500 base pairs must mean that the order of fragments is either 2000–1500–3000 or 3000–1500–2000. If the DNA is circular, the two maps are interchangeable.