A rapid method for cloning and sequencing variable-region genes of expressed immunoglobulins.
A rapid procedure is described for cloning immunoglobulin V region genes from cells that express them. cDNA is synthesized from mRNA template using primers homologous to the immunoglobulin constant-region genes. Blunt-ended, double-stranded cDNA is obtained by sequential addition of enzymes to a single tube. The cDNA is inserted directly into the M13 vector, which is screened by plaque lifting for the presence of specific inserts. Screening probes can be generated from 32P-labeled single-stranded cDNAs generated from primers different from those used for cloning, or alternatively, from previously cloned V or C gene segments. The ease of cloning a cDNA V region is directly related to the abundance of Ig-specific mRNA within the cell of interest. This method minimizes the number of steps and the time needed to obtain accurate and complete sequences of any expressed Ig V region gene.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000378:metabolism; D005796:Genes; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000235:genetics
D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D004247:DNA