B cells are an essential part of the adaptive immune response, functioning via B-cell receptors (BCRs) expressed on their surface. Each B cell expresses a different BCR that allows it to recognize molecular patterns in pathogens. Development of BCRs (Figure 1) is a multistep process in which the progenitor cell undergoes V(D)J recombination in the germline and additional somatic hypermutations (SHM), resulting in a final product with a specific CDR3 (complementarity determining region 3) sequence in the hypervariable region of the immunoglobulin. The unique CDR3 sequence in BCRs is critical for dictating antigen specificity. Taken together, the molecular events described above facilitate receptor diversity and the generation of heavy (H) chain isotypes. BCR diversity enables B cells to recognize and respond to a wide range of pathogens. Upon exposure to a stimulus or stimuli, the lambda (L) and kappa (K) light chain genes of the BCR undergo rearrangements to generate specific gene segments. This further development results in different light chain isotypes being generated from the same B-cell clone.

Figure 1. BCR development. The progenitor cell undergoes recombination of V, D, and J segments in the germline, which generates two identical heavy chains. Recombination of V and J segments generates two identical light chains. Random nucleotide additions or deletions at the junctions of the V, D, and J segments provide additional diversity. Furthermore, B cells activated by immune responses undergo somatic hypermutation (SHM), in which additional point mutations are introduced.
Understanding the profiles of BCRs, (i.e., sequencing the full-length CDR3 regions to determine the diversity of receptors and the clonotypes, defined by expression of specific H, K, and L gene segments) can not only aid in gaining insights into the adaptive immune response in healthy individuals, but also in those with a wide range of conditions, including infectious diseases, allergies, autoimmune disorders, cancers, and aging (Yaari & Kleinstein, 2015). Accurate determination of the clonotypes and isotypes expressed by the immune system will aid in a complete picture of the B-cell repertoire.
Recently, next generation sequencing (NGS) approaches for profiling B-cell repertoires have provided valuable insights into the adaptive immune response and antibody engineering. There are two major approaches used in profiling B-cell repertoires—multiplex PCR and 5' RACE combined with NGS. While multiplexing allows you to amplify multiple BCR genes in one reaction, it may prove challenging with regard to sensitivity, specificity, and biases in amplification of certain sequences, all of which can lead to difficulties in accurate identification of isotypes. On the other hand, the 5'-RACE method reduces variability and allows for priming from the constant region of BCR heavy or light chains. However, the burden of designing optimized primers would still be left to the individual user in this case. The new SMARTer Mouse BCR IgG H/K/L Profiling Kit (SMARTer mouse BCR kit) solves this problem by combining the benefits of 5' RACE with gene-specific amplification (Figure 2) to provide a highly sensitive and reproducible method for profiling B-cell repertoires, by capturing complete V(D)J variable regions of BCR transcripts. The high sensitivity of the kit allows for accurate identification of top clonotypes and reliable assignment of isotype in a majority of cases, based on the sequencing of the H, K, and L chains.
Features of the kit:
- Starts with 10 ng–3 µg of total RNA from spleen, lymph node, PBMCs, and hybridomas
- 5' RACE-based approach combined with gene-specific primer amplification of cDNA libraries is optimized for sensitive and specific clonotype detection
- Optimized library generation workflow for amplification of light chains for mapping and identification of clonotypes
- Accurate amplification of mouse IgG subclasses and identification via sequencing in a majority of cases
The SMARTer Mouse BCR IgG H/K/L Profiling Kit leverages SMART technology (Switching Mechanism at 5' End of RNA Template) and employs a 5' RACE-like approach to capture complete V(D)J variable regions of BCR transcripts. First-strand cDNA synthesis is dT-primed, and the template-switching activity ensures that only sequences from full-length cDNAs undergo PCR amplification (Figure 2). Two rounds of PCR are then performed in succession to amplify cDNA sequences corresponding to the variable regions of BCR IgG heavy chain or BCR light chain (kappa or lambda) transcripts (Figure 2, Panel B). The first PCR uses the first-strand cDNA as a template, and heavy or light chains are amplified in separate reactions. This PCR specifically amplifies the entire variable region and a portion of the constant region of BCR heavy or light chain cDNA. The second PCR uses the first PCR product as a template and uses semi-nested primers to amplify the entire variable region and a portion of the constant region of heavy or light chain cDNA. Following post-PCR purification, size selection, and quality analysis, the library is ready for sequencing on Illumina platforms.

Figure 2. SMARTer Mouse BCR IgG H/K/L Profiling Kit workflow. Panel A. First-strand cDNA synthesis is dT-primed (BCR dT Primer) and performed by the MMLV-derived SMARTScribe Reverse Transcriptase (RT), which adds nontemplated nucleotides upon reaching the 5' end of each mRNA template. The BCR Oligonucleotide anneals to these nontemplated nucleotides and serves as a template for the incorporation of an additional sequence of nucleotides into the first-strand cDNA by the RT (this is the template-switching step). The BCR Oligonucleotide contains sequence from the Illumina Read Primer 2, serving as a primer-annealing site for subsequent rounds of PCR, and ensuring that only sequences from full-length cDNAs undergo amplification. Panel B. The first PCR uses the first-strand cDNA as a template and includes a forward primer with complementarity to the Illumina Read Primer 2 sequence (BCR Primer 1V), and a reverse primer that is complementary to the constant (i.e., nonvariable) region of BCR heavy or light chains (mBCR Primers 1H, 1K, or 1L). The chains are amplified in separate reactions. By priming from the Read Primer 2 sequence and the constant region, the first PCR specifically amplifies the entire variable region and a considerable portion of the constant region of BCR heavy or light chain cDNA. The second PCR takes the product from the first PCR as a template and uses semi-nested primers (mBCR Primers 2H, 2K, or 2L) to amplify the entire variable region and a portion of the constant region of BCR heavy or light chain cDNA. As in PCR 1, the BCR subunit chains are amplified in separate reactions.