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| Modern Genetic Analysis Chapter 10. Recombinant DNA Technology Using Cloned DNA Figure 10-28. Identification of the sequence of restriction-enzyme sites in a circular DNA molecule. In this example, the ring of DNA with a single EcoRI site is opened into a linear molecule by cleavage with EcoRI. The molecule is labeled on its 5 ends with 32P and then partly digested with Alu (restric-tion enzyme from Arthrobacter lutens, which could just as easily be HaeIII, the restriction enzyme from Haemophilus aegyptius) to produce fragments of varying lengths. Each fragment now carries the radioactive label at only one end. The longest fragment is selected (after testing on a gel to compare lengths) and is divided into two samples. One sample is partly digested with Alu, and the other is partly digested with HaeIII. The new sets of fragments are separated on gels. The location of the labeled fragment can be determined by autoradiography of the gels. The sequence of restriction sites can now be read in order as the two gels are compared.

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