Cloning of random oligonucleotides to create single-insert plasmid libraries

Random double-stranded oligonucleotides are useful reagents to identify the optimal binding sites for DNA-binding proteins, such as transcriptional ac tivators. Some applications require ligation of random oligonucleotides to form plasmid-based libraries such as the yeast one-hybrid system, where the activation of a cloned DNA sequence from a library of random DNA-binding s equences activates a reporter gene. Current theories do not account for the low efficiencies of oligonucleotide-based plasmid library construction met hods. We developed a technique to clone single oligonucleotides into plasmi d vectors with high efficiency that predictably results in only one oligonu cleotide insert per colony and used this method to clone a yeast one-hybrid library. This method, either as presented or with modifications, should be suitable for any situation where high-efficiency cloning of single oligonu cleotide inserts is desired. (C) 2001 Academic Press.