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- W4313365622 abstract "Abstract Antibodies are a diverse set of glycoproteins produced by B-cells in response to antigen exposure, infection, or certain malignancies, making it crucial to develop a high throughput and robust technique to obtain paired heavy and light chain information from individual B-cells. We have developed DNA origami-based capture probes to isolate paired heavy and light sequence information from single B-cells. The capture probes are designed to hybridize with the constant regions of heavy and light chains. Each capture probe also incorporates sequences for hybridization with DNA origami nanostructures. The nanostructures can be electroporated into B-cells and re-isolated by cell lysis and streptavidin affinity purification. Heavy and light chains are then reverse transcribed, appending the isolated sequences to the capture probes. Following PCR-amplification, each amplicon will contain BCR heavy or light chain sequence, as well as a unique barcode. Matching barcodes from complementary heavy and light chain amplicons can then be paired via next-generation sequencing. We have used this technique to analyze the antibody repertoire from splenic tissue of unimmunized C57BL/6J mice. We designed a single IgM-Igk specific capture probe and synthesized DNA origami nanostructures. The nanostructures were transfected into naïve B-cells, purified, reverse transcribed, and PCR-amplified. The isolated IgM and Igk amplicons were confirmed by sequencing. Through this approach, we present a novel DNA origami-based technique for single cell paired BCR repertoire analysis. This is a highly versatile technique that can be used to isolate any gene pair functioning in cellular dynamics such as signaling pathways or co-occurring mutations in cancer." @default.
- W4313365622 created "2023-01-06" @default.
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- W4313365622 date "2019-05-01" @default.
- W4313365622 modified "2023-09-26" @default.
- W4313365622 title "DNA origami probes for single-cell paired BCR heavy and light chain immune repertoire analysis" @default.
- W4313365622 doi "https://doi.org/10.4049/jimmunol.202.supp.130.13" @default.
- W4313365622 hasPublicationYear "2019" @default.
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