Plasmid construction by homologous recombination in yeast

Hong Ma, Sam Kunes, Peter J. Schatz, David Botstein

Research output: Contribution to journalArticle

405 Scopus citations

Abstract

We describe a convenient method for constructing new plasmids that relies on interchanging parts of plasmids by homologous recombination in Saccharomyces cerevisiae. A circular recombinant plasmid of a desired structure is regenerated after transformation of yeast with a linearized plasmid and a DNA restriction fragment containing appropriate homology to serve as a substrate for recombinational repair. The free ends of the input DNA molecules need not be homologous in order for efficient recombination between internal homologous regions to occur. The method is particularly useful for incorporating into or removing from plasmids selectable markers, centromere or replication elements, or particular alleles of a gene of interest. Plasmids constructed in yeast can subsequently be recovered in an Escherichia coli host. Using this method, we have constructed an extended series of new yeast centromere, episomal and replicating (YCp, YEp, and YRp) plasmids containing, in various combinations, the selectable yeast markers LEU2, HIS3, LYS2, URA3 and TRP1.

Original languageEnglish (US)
Pages (from-to)201-216
Number of pages16
JournalGene
Volume58
Issue number2-3
DOIs
StatePublished - 1987

All Science Journal Classification (ASJC) codes

  • Genetics

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