Construction of a restriction map

The data of digestion by more than one endonucleases as discussed above can be utilized to arrange the sites of breakage in a defined order. This can be done by several methods, and one of them is illustrated in Figure 40.2. This method involves double digests, where we extract each fragment produced in the individual digests with either enzyme A or enzyme B and then cleave it with the other enzyme. The original DNA sample is also digested by a mixture of both the enzymes to confirm the results of individual successive digests. In Figure 40.3, we have shown the results of such an exercise. When the fragment A-2100 is retrieved and digested with enzyme B, it is cut into two fragments of 1900 bp and 200 bp. Fragment B-2500 (obtained from individual digest with enzyme B) is similarly cut into two fragments of 1900 bp and 600 bp, suggesting overlap of A-2100 and B-2500 (Fig. 40.4), in the region of the fragment of 1900bp which is obtained by one cut enzyme A and the other by enzyme B. It may be seen in Figure 40.2 that in reciprocal digests (A followed by B and B followed by A) same fragments are available. Using this information, we can find the overlapping regions in A and B. digests and find out the sites of cleavage by A and B. This will then allow us to prepare the restriction map as shown in Figure 40.5.