Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction.
A procedure is described for using the polymerase chain reaction (PCR) to amplify and clone the cDNA from mouse immunoglobulin (Ig) variable (V) regions. This method uses a set of universal 5'-oligodeoxyribonucleotide primers that are degenerate and allow for the amplification of Ig V-region sequences from gamma and mu heavy chains and from kappa light chains. Selective first-strand cDNA synthesis is performed using Ig constant region primers and then a PCR is achieved by using the appropriate universal 5'-primer. The universal Ig heavy-chain primer was used to amplify the V-region cDNA from gamma and mu isotypes and the universal light-chain primer was used to amplify three separate kappa light V-region sequences. This procedure was used to obtain Ig V-region gene sequences from hybridomas secreting IgG1/kappa, IgG2b/kappa and IgM/kappa isotypes.
LeBoeuf
R D
RD
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
Galin
F S
FS
Hollinger
S K
SK
Peiper
S C
SC
Blalock
J E
JE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics; D005803:Genes, Immunoglobulin; D006825:Hybridomas / Q000276:immunology; D007132:Immunoglobulin Isotypes / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction
D000911:Antibodies, Monoclonal; D007132:Immunoglobulin Isotypes; D007135:Immunoglobulin Variable Region; D009838:Oligodeoxyribonucleotides; D004247:DNA