Oligonucleotide-mediated, PCR-independent cloning by homologous recombination

We have developed an oligonucleotide-mediated cloning technique based on ho mologous 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 yea st vector by DNA linkers, which consist of two partially overlapping oligon ucleotides. The linkers contain relatively short regions of both yeast vect or 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 allowi ng modification of the transfer-red DNA. Linkers can be designed such that DNA sequences can be transferred with first two reusable universal oligonuc leotides and two gene-specific oligonucleotides. This cloning method, which is performed by co-transforming yeast with linear vector substrate DNA, an d unannealed oligonucleotides, has been termed the yeast-based oligonucleot ide-mediated gap repair technique (YOGRT).