Targeted deletions created in yeast vectors by recombinational excision.
We have developed a simple method for creating defined deletions in yeast vectors by utilizing the ability of Saccharomyces cerevisiae to perform homologous recombination. Two complementary single-stranded oligonucleotides are designed so that the 5' and 3' halves of the resulting double-stranded oligonucleotide are homologous to the 5' and 3' side of a desired deletion junction, respectively. The sequence to be deleted is cleaved by restriction endonuclease digestion, followed by co-transformation of the linearized plasmid and the oligonucleotide into yeast. By homologous recombination in vivo, a subset of the plasmids will recircularize and simultaneously acquire the deletion as defined by the oligonucleotide.
Dunø
M
M
Department of Animal Breeding and Genetics, Danish Institute of Agricultural Sciences, PO Box 50, DK-8830 Tjele, Denmark.
Bendixen
C
C
Krejci
L
L
Thomsen
B
B
D004262:DNA Restriction Enzymes / Q000378:metabolism; D005822:Genetic Vectors; D009841:Oligonucleotides / Q000737:chemistry; D010957:Plasmids; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017384:Sequence Deletion; D014170:Transformation, Genetic
D009841:Oligonucleotides; D004262:DNA Restriction Enzymes