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Methods Enzymol. 2010;470:181-204. doi: 10.1016/S0076-6879(10)70008-2. Epub 2010 Mar 1.

Making temperature-sensitive mutants.

Author information

1
Michael Smith Laboratories, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

Abstract

The study of temperature-sensitive (Ts) mutant phenotypes is fundamental to gene identification and for dissecting essential gene function. In this chapter, we describe two "shuffling" methods for producing Ts mutants using a combination of PCR, in vivo recombination, and transformation of diploid strains heterozygous for a knockout of the desired mutation. The main difference between the two methods is the type of strain produced. In the "plasmid" version, the product is a knockout mutant carrying a centromeric plasmid carrying the Ts mutant. In the "chromosomal" version, The Ts alleles are integrated directly into the endogenous locus, albeit not in an entirely native configuration. Both variations have their strengths and weaknesses, which are discussed here.

PMID:
20946811
PMCID:
PMC2957654
DOI:
10.1016/S0076-6879(10)70008-2
[Indexed for MEDLINE]
Free PMC Article

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