A small DNA fragment can be randomly mutagenized in vitro by exposing it to a chemical mutagen. The DNA fragment can then be moved into an appropriate recipient cell by taking advantage of a linked selectable genetic marker. This approach allows localized random mutagenesis of a small, specific region of DNA without producing secondary mutations elsewhere on the chromosome. Such localized mutagenesis is especially useful for obtaining rare point mutations in or near a gene of interest (for example, temperature-sensitive mutations in a gene, mutations in the promoter or operator of a gene, or mutations that affect amino acids at the active site of an enzyme).
Hydroxylamine is a very useful mutagen for localized mutagenesis. Unlike many mutagens, hydroxylamine can mutagenize DNA packaged inside of phage heads. This allows the mutagenesis of transducing particles in vitro (Hong and Ames, 1971).
Localized mutagenesis of transducing DNA with hydroxylamine involves several steps. (1) A phage lysate containing transducing particles is mutagenized with hydroxylamine in vitro. The extent of mutagenesis of the phage particles can be monitored indirectly by following decrease in phage titer (or "killing") due to mutations in essential phage genes, or directly by following the increase in clear plaque phage mutants in the lysate. (2) Recipient cells are then transduced with the mutagenized lysate, selecting for a specific marker carried by the transducing particle. When the transducing fragment is recombined onto the chromosome, only the small, localized region carried on that mutagenized transducing fragment is inherited. (3) The resulting transductants are then screened for point mutations that affect the linked gene (that is, the "unselected marker). The frequency of such mutations will depend upon how heavily the transducing lysate was mutagenized and how closely linked the selected and unselected markers are.
Essentially any marker with a selectable phenotype can be used for localized mutagenesis. For example, the selected marker can repair an auxotrophic mutation as shown in the figure below.
However, many genes do not have closely linked, easily selectable genetic markers. In these cases a linked transposon can be used as the selectable marker. For example, a transducing lysate grown on a strain with a Tn10 insertion closely linked to some particular gene can be used to bring the linked gene into a recipient cell by selection for the antibiotic resistance (TetR) encoded by Tn10. Using this trick, it is possible to do localized mutagenesis of essentially any region of the chromosome. An example is shown in the figure below.
In addition to mutagenesis of DNA in phage particles, this is also an effective method for isolating mutations in purified plasmid DNA.
Please send comments, suggestions, or questions to email@example.com
Last modified October 14, 2003