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 activators. 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 sequences activates a reporter gene. Current theories do not account for the low efficiencies of oligonucleotide-based plasmid library construction methods. We developed a technique to clone single oligonucleotides into plasmid vectors with high efficiency that predictably results in only one oligonucleotide 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 oligonucleotide inserts is desired.
Worthington
M T
MT
The Digestive Health Research Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA. mtw3p@virginia.edu
Pelo
J
J
Luo
R Q
RQ
D003001:Cloning, Molecular / Q000379:methods*; D015723:Gene Library; D009841:Oligonucleotides / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
D009841:Oligonucleotides