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1. 1 2 3 4 5 Agarose gel electrophoresis/ Restriction maps Genial- Worksheet IX.10 Particular DNA fragments can be isolated by cutting out the small region of the gel that contains the fragment and removing the DNA from the gel. One important use of isolated restriction fragments employs the enzyme DNA ligaseto insert them into self-replicating molecules such as bacteriophage, plasmids, or even small artificial chromosomes. These procedures constitute DNA cloning and are the basis of one form of genetic engineering (Brown 1991). DNA fragments that have been cloned into organisms such as E. coli are widely used because the fragments can be isolated in large amounts and purified relatively easily. Among the uses of cloned DNA are: · DNA sequencing. All current methods of DNA sequencing require cloned DNA fragments. · Nucleic acid hybridisation. As we shall see, an important application of cloned DNAfragments entails incorporating a radioactive or light-emitting "label" into them, after which the labelled material is used to "tag" DNA fragments containing similar sequences. · Storage and distribution. Cloned DNA can be stored for long periods without risk of change and can easily be distributed to other researchers (Hartl and Jones 2000). option: Restriction maps Because of the sequence specificity of cleavage, a particular restriction enzyme produces a unique set of fragments for a particular DNA molecule. Another enzyme will produce a different set of fragments from the same DNA molecule. In Figure 1, this principle is illustrated for the digestion of lDNA by either Xba I, Xho I or Kpn I. The locations of the cleavage sites for these enzymes in lDNA are shown in Figure 1 (b). A diagram showing sites of cleavage along a DNA molecule is called a restriction map. (a) (b) Figure 1.l DNA was digested with the enzymes Xba I, Xho I and Kpn I in single and double digestions. a) stained gel b) construction of a restriction map (Brown 1991) page: