Cloning of random oligonucleotides to create single-insert plasmid libraries.

Autor: Worthington MT; The Digestive Health Research Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA. mtw3p@virginia.edu, Pelo J, Luo RQ
Jazyk: angličtina
Zdroj: Analytical biochemistry [Anal Biochem] 2001 Jul 15; Vol. 294 (2), pp. 169-75.
DOI: 10.1006/abio.2001.5162
Abstrakt: 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.
(Copyright 2001 Academic Press.)
Databáze: MEDLINE
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