Figure 5-12. Diagrammatic representation of gene cloning experiment.

Figure 5-12Diagrammatic representation of gene cloning experiment

Plasmid cloning vector pBR322 is 4.36 kilobase pairs in size, has genes for resistance to ampicillin (ampr) and to tetracycline (tetr), and has only one HindIII restriction site that is located within the tetr locus. HindIII is used to treat samples of DNA from plasmid pBR322 and from a donor organism with a gene, designated a+, to be cloned. The donor can be a prokaryotic or a eukaryotic organism. If HindIII restriction sites are located adjacent to a+ in donor DNA, but do not occur within a+, a restriction fragment carrying intact a+ marker can be generated from donor DNA. Hybrid plasmids can be formed by random association and ligation of the HindIII-treated donor and vector DNA fragments. Although pBR322 is tetr, hybrid plasmids will be tets because the donor DNA fragments are inserted at the HindIII restriction site within the tetr locus. After transformation of amps-recipient bacteria that also lack a+, transconjugants with hybrid plasmids can be selected by their ampr tets phenotypes. Strains in which the a+ gene is present can then be identified by expresion of a+ or by testing for the polynucleotide sequence corresponding to a+. The pBR322 plasmid contains other uniuqe restriction sites that can also be used for cloning (e.g., PstI in ampr and BamHI in tetr). Many other cloning vectors and restriction endonucleases have also been used for gene cloning experiments.

From: Chapter 5, Genetics

Cover of Medical Microbiology
Medical Microbiology. 4th edition.
Baron S, editor.
Copyright © 1996, The University of Texas Medical Branch at Galveston.

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