Walking Along a Chromosome to Clone a Gene

Walking Along a Chromosome to Clone a Gene

Another method of cloning a desired gene is walking. Suppose a randomly isolated clone has been shown to lie within several hundred thousand bases of the DNA segment we wish to clone. Of course, demonstrating such a fact is not trivial in itself. In the case of the fruit fly Drosophila melanogaster, however, the demonstration is straightfor-ward.

Figure 9.14 (a) A gel showing DNA fragments resulting from a restrictionenzyme digestion of a set of four partially overlapping DNA sequences. (b) The four DNAs and their composite.

As explained earlier, the polytene chromosomes in cells of several Drosophila tissues possess characteristic bands that serve as chromosomal landmarks. Therefore the DNA involved in chromosome rearrange-ments or deletions can readily be observed. Also, highly radioactive DNA originating from a cloned segment of Drosophila chromosome can be hybridized in situ to Drosophila polytene chromosomes. The position to which the fragment hybridizes can then be determined by autoradiog-raphy. Hence the chromosomal position of a cloned fragment can be determined. If it lies near a gene of interest, walking may be in order.

A chromosomal walk to clone a specific gene begins with a restriction map of the cloned piece of DNA. The right- and left-end terminal restriction fragments are used as probes of a lambda bank. Several clones are picked that hybridize to the right-hand fragment and not to the left-hand fragment. Then restriction maps of these clones are made and again the right- and left-hand fragments are used to find new clones that hybridize only to the right-hand fragments (Fig. 9.14). The succes-sive lambda transducing phage that are identified permit walking to the right, and when a sufficient distance has been covered, in situ hybridi-zation permits determination of which direction on the cloned DNA corresponds to moving toward the desired gene.