Cloning of Random Oligonucleotides to Create Single-Insert Plasmid Libraries
| Autor: | Mark T. Worthington, Roger Qi Luo, Jared W. Pelo |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Plasmid preparation
clone (Java method) Genetics Cloning Reporter gene Library Oligonucleotide Oligonucleotides Biophysics Cell Biology Computational biology Biology Biochemistry Insert (molecular biology) Plasmid Two-Hybrid System Techniques Yeasts Cloning Molecular Molecular Biology Gene Library Plasmids |
| Zdroj: | Analytical Biochemistry. 294:169-175 |
| ISSN: | 0003-2697 |
| DOI: | 10.1006/abio.2001.5162 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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