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.
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.
Schoonbroodt
Sonia
S
Dyax s.a., Boulevard du Rectorat 27B Sart Tilman, B-4000 Liege 1, Belgium.
Frans
Nicolas
N
DeSouza
Mark
M
Eren
Rachel
R
Priel
Smadar
S
Brosh
Naama
N
Ben-Porath
Judith
J
Zauberman
Arie
A
Ilan
Ehud
E
Dagan
Shlomo
S
Cohen
Edward H
EH
Hoogenboom
Hennie R
HR
Ladner
Robert Charles
RC
Hoet
René M
RM
D000293:Adolescent; D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001709:Biotechnology / Q000379:methods; D002176:Candida albicans / Q000201:enzymology / Q000276:immunology; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D005260:Female; D005803:Genes, Immunoglobulin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008875:Middle Aged; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D009841:Oligonucleotides; D019151:Peptide Library; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases