Restriction cleavage and gel electrophoresis
If we take a particular DNA molecule or DNA sample, digest it with a specific restriction enzyme and then subject the sample to gel electrophoresis (a technique in which DNA digest is loaded on gel slab and the DNA fragments are allowed to move under the influence of an electric current), we will notice a series of bands on the gel slab or cylinder. The position of different bands will depend on DNA fragment size, such that smaller the fragment, more rapidly it will move, and longer the fragment, more slowly will it move. It will mean that the fragment away from the loading site will be smaller and those close to the loading site will represent longer DNA fragments. The gel can be calibrated by using a mixture of DNA fragments of known lengths so that the position of bands on this standard gel can be compared with the bands in the experimental DNA digest and the fragment length in each band of DNA digest can thus be determined. In Figure 40.1, we have shown the results of digestion of 5000 bp long DNA molecule digested separately by two enzymes A and B. As shown in the figure, the enzyme A cleaves the DNA into four fragments of lengths 2100, 1400, 1000 and 500 bp, while the enzyme B cleaves it into three fragments of length 2500, 1300, 1200 bp. These data can be used to generate a map.