Saccharomyces_A

Incubate gene replacement creates directed mutations

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Unformatted text preview: ic kanamycin. Incubate Gene Replacement Creates Directed Mutations Gene replacement is a powerful technique that has a great advantage over classical methods of strain construction: Researchers can use gene replacement to alter at will a chromosomal gene in a living cell without changing the genome in any other way. This level of control over gene alteration makes it possible to compare the phenotypic effects of mutations in an otherwise isogenic (that is, genetically identical) background and attribute any phenotypic differences to specific alleles. The technique of gene replacement exploits the high level of homologous recombination in yeast. Linear fragments that contain beginning and ending sequences of a gene with a selectable marker cloned between the two gene parts are transformed into yeast and recombinants are selected. Figure A.8 illustrates gene replacement via transformation Cells with disrupted gene are KANR and grow. Figure A.8 Gene disruption and replacement. A gene is disrupted by first constructing a linear fragment in which the KAN R gene (to be used as a selectable marker) replaces the middle of the gen...
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