Restriction Mapping of DNA Fragments 

Restriction mapping involves the size analysis of restriction fragments produced by several restriction enzymes individually and in combination. The principle of this mapping is illustrated, in which the restriction sites of two enzymes, A and B, are being mapped. Cleavage with A gives fragments 2 and 7 kilobases (kb) from a 9 kb molecule, hence we can position the single A site 2 kb from one end. Similarly, B gives fragments 3 and 6 kb, so it has a single site 3 kb from one end; but it is not possible at this stage to say if it is near to A’s site or at the opposite end of the DNA.  This can be resolved by a double digestion. If the resultant fragments are 2, 3 and 4 kb, then A and B cut at opposite ends of the molecule; if they are 1, 2 and 6 kb, the sites are near each other. Not surprisingly, the mapping of real molecules is rarely as simple as this and computer analysis of the restriction fragment lengths is usually needed to construct a map (Figure 1).

Figure 1. Restriction fragment length polymorphisms (RFLP). The schematic panels A–F indicate the various fragments obtained following digestion as a result of differences in the position of restriction endonuclease target sequences.