Oligonucleotide-mediated, PCR-independent cloning by homologous recombination

We have developed an oligonucleotide-mediated cloning technique based on homologous recombination in Saccharomyces cerevisiae that allows precise DNA sequences to be transferred independent of restriction enzymes and PCR. In this procedure, linear DNA sequences are targeted to a chosen site in a yeast vector by DNA linkers, which consist of two partially overlapping oligonucleotides. The linkers contain relatively short regions of both yeast vector sequences and insert sequences, which stimulate homologous recombination between the vector and the insert. The linkers cart also contain sequences not found in either the vector or the insert (e.g., sequences that encode ribosome binding sites, epitope tags, prefer red codons, etc.), thus allowing modification of the transfer-red DNA. Linkers can be designed such that DNA sequences can be transferred with first two reusable universal oligonucleotides and two gene-specific oligonucleotides. This cloning method, which is performed by co-transforming yeast with linear vector substrate DNA, and unannealed oligonucleotides, has been termed the yeast-based oligonucleotide-mediated gap repair technique (YOGRT).