Figure 18-28 Cloning a Gene in the Plasmid Vector pUC19

Chapter 18

Sexual Reproduction, Meiosis, and Genetic Recombination

Figure 18-28 Cloning a Gene in the Plasmid Vector pUC19

(b) Insertion of foreign DNA into the plasmid. (1) The plasmid is cleaved with a restriction enzyme known to recognize a single site, in this case one that is within the lacZ gene. (2) The same enzyme is used to cleave a foreign DNA molecule containing a gene of interest, thereby generating fragments that have the same cohesive ends as the linearized plasmid DNA. (3) The fragments of foreign DNA are then incubated with the linearized plasmid DNA under conditions that favor base pairing between cohesive ends. Among the expected products will be plasmid molecules recircularized by base pairing with a single fragment of foreign DNA, and some of these will contain the gene of interest. (4) Incubation with DNA ligase covalently seals such a recombinant molecule, generating a recombinant plasmid vector carrying the gene of interest. Cells carrying such plasmids will be resistant to ampicillin and will fail to produce beta galactosidase because of the foreign DNA inserted within the lacZ gene.

© 1999 by Addison Wesley Longman
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