Strategies for gene disruptions and plasmid constructions in fission yeast.
Molecular genetic analyses in Schizosaccharomyces pombe are greatly enhanced by our ability to delete chromosomal genes via homologous recombination and to introduce genes expressed from autonomous plasmids. In this paper, we describe a novel approach to generating marked deletion cassettes that bypasses the need for the long, PAGE-purified oligonucleotides required in the currently used PCR-based deletion approach. We also describe additional uses of this two-step PCR method for constructing chromosomal insertion cassettes. Finally, we describe how gap repair in S. pombe can facilitate plasmid constructions in a manner that circumvents the reliance on compatible restriction sites in the DNA molecules that are being joined. Several applications of this gap repair plasmid construction strategy are discussed.
Wang
Lili
L
Boston College, Biology Department, Higgins Hall 401B, Chestnut Hill, MA 02467, USA.
Kao
Richard
R
Ivey
F Douglas
FD
Hoffman
Charles S
CS
D017353:Gene Deletion; D016254:Mutagenesis, Insertional / Q000379:methods; D010957:Plasmids / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics*