This kit provides the specific primers required for the amplification of mouse antibody variable regions (Gamma, Kappa, and IgG heavy chains) from hybridoma total RNA. The workflow involves:
Reverse Transcription: RNA is converted to cDNA using primers that bind conserved constant-region sequences. An adapter sequence is introduced during this step to provide a standardized priming site for subsequent amplification.
PCR Amplification: The resulting cDNA is amplified using a forward primer complementary to the adapter and reverse primers specific to the antibody chain type. A nested PCR design improves specificity and product yield.
Sequencing: The resulting ~600-850bp amplicons are purified for Sanger sequencing to determine the variable region DNA sequence.
This kit is for sequencing the variable regions of human monoclonal IgG antibodies (subclasses IgG1 to IgG4) from RNA sources such as recombinant expression cell lines, hybridomas, or single B cells. Using an adapter-based cDNA synthesis approach, the kit now provides complete light chain coverage for both kappa and lambda chains, amplifying IgG heavy chains and kappa light chains to ~750 to 850 bp, and lambda light chains to ~600 to700 bp with newly integrated human-specific constant region primers. Key limitations include restriction to IgG isotypes, lack of validation for engineered antibodies with altered constant regions or low RNA integrity, and the inability to perform repertoire analysis from polyclonal samples without prior single-cell isolation.
