Optimization of primers for cloning libraries of mouse immunoglobulin genes using the polymerase chain reaction.
We have optimized primers for cloning libraries of murine heavy and light chain variable regions using the polymerase chain reaction. Since we are interested in cloning murine Fab fragments for expression in bacterial cells, the heavy chain primers were designed to clone Fd fragments comprising the heavy chain variable domain and the first domain of the IgG constant region. The light chain primers were designed to clone the entire murine kappa chain. Using ten degenerate 5' primers and a degenerate 3' primer to amplify murine Fd and seven degenerate 5' primers with a single 3' primer to amplify kappa chains, a diverse repertoire of mouse variable regions was cloned from mouse spleens.
Kettleborough
C A
CA
MRC Collaborative Centre, London, GB.
Saldanha
J
J
Ansell
K H
KH
Bendig
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015723:Gene Library; D005803:Genes, Immunoglobulin; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains