Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction.
A general method to directly obtain the DNA sequence of the variable regions of any immunoglobulin chain using a mixture of oligomer primers and the polymerase chain reaction (PCR) is described. Mixed oligonucleotide primers corresponding to the 5' signal peptide and a conserved 3' constant region primer were used for enzymatic amplification of each of the heavy and light chain variable regions of a human hybridoma producing a monoclonal antibody recognizing an epitope of gp120 of the human immunodeficiency virus 1. The amplified DNA segments were cloned and the sequence was determined for the heavy chain variable region. This method will greatly facilitate structural and functional studies of immunoglobulins by reducing the effort to clone and sequence the members of the immunoglobulin as well as other multigene families.
Larrick
J W
JW
Genelabs Incorporated, Redwood City, CA 94063.
Danielsson
L
L
Brenner
C A
CA
Abrahamson
M
M
Fry
K E
KE
Borrebaeck
C A
CA
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005784:Gene Amplification; D005803:Genes, Immunoglobulin; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D009841:Oligonucleotides; D012333:RNA, Messenger; D004247:DNA; D004259:DNA-Directed DNA Polymerase