UNIT 13.10 Manipulation of Cloned Yeast DNA
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Lundblad, V., Hartzog, G. and Moqtaderi, Z. 2001. Manipulation of Cloned Yeast DNA. Current Protocols in Molecular Biology. 39:III:13.10:13.10.1–13.10.14.
- Published Online: 1 MAY 2001
- Published Print: JUL 1997
A major advantage of working with yeast is the ability to replace the wild-type chromosomal copy of a gene with a mutant derivative that is constructed in vitro using a cloned copy of the gene. This techniqueunavailable in most other eukaryotesallows the phenotype of the mutation to be studied under accurate in vivo conditions, with the mutation present in single copy at its normal chromosomal location. In the first protocol, a plasmid harboring both a selectable marker and a cloned gene of interest is integrated at the chromosomal location of the cloned gene via homologous recombination (integrative trandformation). Four methods are described for constructing a mutation in vitro in a cloned gene and reintroducing this mutation at the correct chromosomal site. This allows assessment of the genetic consequences of a mutation, and is often used to determine whether or not a gene is essential (by determining if a complete gene deletion is viable). Two of these techniquesintegrative disruption and one-step gene disruptiongenerate either insertion or deletion mutations. The third techniquetransplacementis more generally applicable: it can be used to introduce insertion or deletion mutations containing a selectable marker, but it can also be used to introduce nonselectable mutations, such as conditional lethal mutations in an essential gene. Protocols are also provided to allow creation of modified genes by one-step integrative replacement, and also conditional alleles by a copper-inducible double-shutoff procedure.