532 Chapter 16 • Dissection of Gene Function mutations in the resident gene [Figure 16-16a(ii) and (iii)]. The oligonucleotide-directed method can also be used on genes cloned in double-stranded vectors if their DNA is ±rst denatured. A knowledge of restriction sites is also useful in di-rected mutation of a transgene. For example, a small dele-tion can be made by removing the fragment that is liber-ated by cutting at two restriction sites (Figure 16-16b). With a similar double cut, a fragment, or cassette, can be inserted at a single restriction cut to create a duplication or other modi±cation (Figure 16-16c). Another approach is to erode enzymatically a cut end created by a restric-tion enzyme to create deletions of various lengths (Figure 16-16d). PCR also can be used to generate a DNA frag-ment containing a speci±c mutation, for eventual intro-duction as a transgene (Figure 16-16e). In the fungus
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This note was uploaded on 01/10/2011 for the course BIOL BIOL taught by Professor Johnson during the Spring '08 term at Aberystwyth University.