6.2 DNA Mapping

A prerequisite condition for sequencing high-molecular weight DNAs is a preparation of a restriction map - that is, representing the mutual arrangement of sites of cleavage by particular restriction endonucleases over the DNA fragment of interest and approximate distances between them.

The mutual arrangement of the large blocks resulting from the cleavage of the DNA chain by restriction enzymes can be determined without any prior information about the entire sequence of nucleotides in these blocks. This is possible owing to the specific action of restriction enzymes. One can resort for this purpose both to incomplete cleavage by the same restriction enzyme and to another restriction enzyme. The method is based on the sharp difference between the molecular weights of the fragments resulting from complete and incomplete cleavage, as well as the possibility of distinct separation of the cleavage products by means of electrophoresis. The latter is performed in a polyacrylamide or agarose gel. What happens in the process is separation of DNA fragments by molecular weight. Having plotted mobility versus molecular weight (for which purpose low-molecular weight DNAs of some phages with a known molecular weight are employed), one can find the molecular weight of the DNA under analysis from its electrophoretic mobility.

An example of such an approach is provided by the following scheme of analysis of SV40 virus DNA with a molecular weight of 3 ^. 10⁶.

Complete cleavage of this DNA, which is essentially a cyclic double-stranded polydeoxyribonucleotide, by a mixture of restriction enzymes Hind isolated from Haemophilus influenzae R_d gives 11 fragments equal in length to 400 to 2500 nucleotides; they are shown on the diagram as fragments A through K. They are separated electrophoretically in polyacrylamide gel. At the same time, the initial DNA is treated with the same enzyme under mild conditions to obtain, then to separate by the same method, polynucleotides with a molecular weight higher than in the first case. After complete cleavage of these blocks the investigator determines which of the fragments A through K they contain and in what sequence (see Fig. 6-1).

By using the overlapping block method one can determine the structure of the DNA of interest. The data for the SV40 virus DNA are shown in Table 6-1.

Table 6-1. Results of Cleaving SV40 Virus DNA by a Mixture of Restriction Endonucleases Hind.

* The fractions are arranged in decreasing order of their mobility during PAGE.

It can be seen that the fragments occur in the following sequence:

The position of fragments C and D as well as H and I cannot be determined from the above data. The location of all fragments was determined by using a mixture of other restriction enzymes Hpa (see above), which cleave SV40 DNA into three fragments: Hpa A, Hpa B and Hpa C with chains 4200, 3400 and 2000 nucleotides long, respectively, each fragment being subsequently treated by a mixture of enzymes Hind. Under the action of the latter, Hpa A was cleaved into fragments D, E, F, G, J, K and a portion of C; Hpa B was cleaved into fragments A, H and a portion of C; and Hpa C was cleaved into fragments B and I. By comparing these results one can distribute all eleven fragments A through K as shown in the scheme. The results also make it possible to define the mutual arrangement of the three fragments produced by the Hpa-mediated cleavage.

The next section covers some restriction enzymes widely used in genetic engineering, including DNA mapping and fragmentation.