Cloning of random oligonucleotides to create single-insert plasmid libraries

Mark T. Worthington, Jared Pelo, Roger Qi Lo

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)169-175
Number of pages7
JournalAnalytical biochemistry
Issue number2
StatePublished - Jul 15 2001
Externally publishedYes


  • Histidine prototrophy
  • Ligation
  • One-hybrid screening
  • Saccharomyces cerevisiae

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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