Oligonucleotide-directed site-specific integration of high complexity libraries into ssDNA templates

Nucleic Acids Res. 2004 Jan 29;32(2):e22. doi: 10.1093/nar/gnh021.

Abstract

We present an approach that generates an oligomer-based library with minimal need for restriction site modification of sequences in the target vector. The technique has the advantage that it can be applied for generating peptide aptamer libraries at sites within proteins without the need for introducing flanking enzyme sites. As an example we present a phagemid retroviral shuttle vector that can be used to achieve stable expression of the library in mammalian cells for the purpose of screening for peptides with desired biological activity.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Binding Sites / genetics
  • Cell Line
  • Cell Survival / drug effects
  • Cell Survival / genetics
  • Cloning, Molecular / methods
  • DNA / genetics
  • DNA / metabolism
  • DNA, Single-Stranded / genetics*
  • DNA, Single-Stranded / metabolism
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Escherichia coli / genetics
  • Gene Library*
  • Genetic Vectors / genetics
  • Humans
  • Molecular Sequence Data
  • Oligonucleotides / genetics*
  • Pyrrolidinones / pharmacology
  • Reproducibility of Results
  • Retroviridae / genetics
  • Templates, Genetic
  • Transfection

Substances

  • DNA, Single-Stranded
  • Oligonucleotides
  • Pyrrolidinones
  • blasticidin A
  • DNA
  • endodeoxyribonuclease PaeI
  • CTCGAG-specific type II deoxyribonucleases
  • Deoxyribonucleases, Type II Site-Specific