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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
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This note was uploaded on 10/03/2013 for the course BI 206 taught by Professor Celenza during the Spring '08 term at BU.
- Spring '08