![]() The emulsion PCR protocol based on ABIL EM 90 surfactant was adapted from 20, with revisions to allow for single-stage, cell-based RT and amplification reactions. We employed a strategy of starting from RNA, which benefits from a high abundance of molecules encoding TCR alpha and beta chains and universal primer annealing sites on the constant regions of the TCR chains 18, 19. (D) Data shown are representative of three experiments performed. PCR suppression (lanes 4–6) eliminated random overlap extension efficiently compared to reactions performed without such suppression (lanes 1–3). (D) Postemulsion amplification of fused TCR molecules using: (lanes 1, 4) product of emulsion reaction that initially contained all primers from the start (αβ), (lanes 2, 5) combined products of two separate emulsions, each initially containing only primers for either TCR alpha or beta amplification (as a control for random overlap extension after emulsion), and (lanes 3, 6) combined product of two separate emulsions that initially contained primers for either alpha or beta TCR amplification but were joined after RT step (as a control for random overlap extension due to fusion of cell-containing emulsion droplets during PCR). (C) Reaction products are extracted from the emulsion and fused molecules of interest are selectively amplified, while nonfused molecules are suppressed by blockers. (B) Released TCR alpha and beta mRNAs are reverse-transcribed at 50☌ with specific primers, amplified, and overlap extended within each droplet. 1A).Ĭell-based emulsion RT-PCR technique for identifying TCR alpha–beta chain pairing. The key requirement for the success of our approach is the ability to generate a representative library of TCR alpha and beta genes that are fused within emulsion reactions, wherein individual T cells are each contained within a separate minute droplet of sufficient reaction volume (Fig. Results and discussion RT and amplification from cells in emulsion Notably, the whole technology is simple to implement and does not require any special equipment. We overcame the primary obstacle of random, nonspecific overlap extension during postemulsion amplification reactions with a new PCR-suppression technique that we invented to specifically and efficiently block the 3′-ends of the free, nonoverlapped alpha and beta chain PCR products. Here, we report a new approach for identifying TCR alpha–beta pairing, based on specific RT of TCR alpha and beta chain mRNA, PCR amplification, and subsequent coupling via overlap extension 17, all performed within emulsion droplets each of which contains a single T cell. However, this assay is quite laborious and requires the fabrication of custom chips, while its efficiency is not fully clear. Recently, a micro-well plate assay was reported to pair human immunoglobulin heavy and light chains for high-throughput sequencing 16. Furthermore, those approaches that could yield massive output based on the amplification and assembly of genes in fixed cells 14, 15 have not demonstrated feasibility for the analysis of complex samples. Phage and yeast display technologies 11- 13, although efficient for isolation of antigen-specific antibodies, rely on random pairing and do not provide information on the native pairs of chains. While these approaches are feasible to identify native chain pairs, they can only be performed serially for a limited number of clones, thereby prohibiting comprehensive analysis of most biological samples. Native pairs of TCR or antibody chains can also be identified after culturing of lymphocyte clones 5, or sorting of narrow antigen-specific populations of T cells 9 or B cells 10. Because of this, methods for efficient identification of their functional units - native pairs of heavy/light antibody or alpha/beta TCR chains - have been highly desired since decades.Ī series of different approaches have been developed to this end, including hybrid cells 4, single-cell PCR 5- 7, and frequency-based pairing 8. Analysis of the native TCR and antibody repertoires is also of fundamental importance for our understanding of adaptive immunity in health and disease 3. ![]() Antibodies and T-cell receptors (TCRs), the weapons of adaptive immunity capable of selective recognition of specific antigens, represent an invaluable resource for biological studies and medical applications 1, 2.
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