TY - JOUR
T1 - Cloning of random oligonucleotides to create single-insert plasmid libraries
AU - Worthington, Mark T.
AU - Pelo, Jared
AU - Lo, Roger Qi
N1 - Funding Information:
We are grateful to Phil Hieter for his gift of yeast strain YPH499 and plasmid pRS314, to Jeffery Milbrandt for plasmid pHR307a, and to Gregory Kato for helpful discussions. This work was supported in part by NIH Grant DK02501 and by the American Digestive Health Foundation Industry Research Scholar Award (to M.T.W.).
PY - 2001/7/15
Y1 - 2001/7/15
N2 - 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.
AB - 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.
KW - Histidine prototrophy
KW - Ligation
KW - One-hybrid screening
KW - Saccharomyces cerevisiae
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U2 - 10.1006/abio.2001.5162
DO - 10.1006/abio.2001.5162
M3 - Article
C2 - 11444813
AN - SCOPUS:0035879501
SN - 0003-2697
VL - 294
SP - 169
EP - 175
JO - Analytical biochemistry
JF - Analytical biochemistry
IS - 2
ER -