Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library

Nucleic Acids Res. 2005 May 19;33(9):e81. doi: 10.1093/nar/gni080.

Abstract

The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Adolescent
  • Adult
  • Animals
  • Antibodies, Monoclonal / immunology
  • Antibody Specificity
  • Biotechnology / methods
  • Candida albicans / enzymology
  • Candida albicans / immunology
  • Cloning, Molecular / methods*
  • DNA, Complementary*
  • Female
  • Genes, Immunoglobulin*
  • Glyceraldehyde-3-Phosphate Dehydrogenases / immunology
  • Humans
  • Immunoglobulin Fab Fragments / chemistry
  • Immunoglobulin Fab Fragments / immunology
  • Immunoglobulin Fab Fragments / isolation & purification
  • Immunoglobulin Heavy Chains / chemistry
  • Immunoglobulin Heavy Chains / genetics
  • Immunoglobulin Variable Region / genetics
  • Mice
  • Mice, Inbred BALB C
  • Middle Aged
  • Oligonucleotides / chemistry*
  • Oligonucleotides / metabolism
  • Peptide Library*
  • Polymerase Chain Reaction
  • Sequence Analysis, DNA

Substances

  • Antibodies, Monoclonal
  • DNA, Complementary
  • Immunoglobulin Fab Fragments
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Variable Region
  • Oligonucleotides
  • Peptide Library
  • Glyceraldehyde-3-Phosphate Dehydrogenases