Rapid and Efficient Plasmid Construction by Homologous Recombination in Yeast.
The cloning of DNA fragments is a fundamental aspect of molecular biology. Traditional DNA cloning techniques rely on the ligation of an insert and a linearized plasmid that have been digested with restriction enzymes and the subsequent introduction of the ligated DNA into Escherichia coli for propagation. However, this method is limited by the availability of restriction sites, which often becomes problematic when cloning multiple or large DNA fragments. Furthermore, using traditional methods to clone multiple DNA fragments requires experience and multiple laborious steps. In this protocol, we describe a simple and efficient cloning method that relies on homologous recombination in the yeast Saccharomyces cerevisiae to assemble multiple DNA fragments, with 30-bp homology regions between the fragments, into one sophisticated construct. This method can easily be extended to clone plasmids for other organisms, such as bacteria, plants, and mammalian cells.
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada;
Jolanda
J
van Leeuwen
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
Brenda
B
Andrews
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada.
Charles
C
Boone
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada;
Guihong
G
Tan
D003001:Cloning, Molecular / Q000379:methods*; D059765:Homologous Recombination; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000235:genetics*