The use of the RACE method to clone hybridoma cDNA when V region primers fail.
The technique of V region PCR to clone antibody V regions from hybridomas has been extensively used. However, in addition to, or even instead of, cloning the V regions with the desired specificity, myeloma cell derived V regions, V regions which are the result of non-productive rearrangements, and also V regions which are productive but which do not recognise the antigen of interest, may be isolated. In this paper we describe a comparison of the use of V region PCR and a modification of the RACE technique to clone the V region of the anti-NGF hybridoma, alpha D11. This hybridoma has heavy and light chain V regions which are refractory to amplification with V region primers, but which are easily amplified using RACE, a PCR based procedure which is independent of the variability within the V regions.
Ruberti
F
F
International School for Advanced Studies (SISSA), Trieste, Italy.
Cattaneo
A
A
Bradbury
A
A
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D005784:Gene Amplification; D006825:Hybridomas / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007158:Immunologic Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009414:Nerve Growth Factors / Q000276:immunology; D016133:Polymerase Chain Reaction; D051381:Rats
D017931:DNA Primers; D018076:DNA, Complementary; D007135:Immunoglobulin Variable Region; D009414:Nerve Growth Factors