Sequencing heavy- and light-chain variable genes of single B-hybridoma cells by total enzymatic amplification.
نویسندگان
چکیده
منابع مشابه
Specific amplification by PCR of rearranged genomic variable regions of immunoglobulin genes from mouse hybridoma cells.
We have designed a novel strategy for the isolation of the rearranged genomic fragments encoding the L-VH-D-JH and L-V kappa/lambda-J kappa/lambda regions of mouse immunoglobulin genes. This strategy is based on the PCR amplification of genomic DNA from mouse hybridomas using multiple specific primers chosen in the 5'-untranslated region and in the intron downstream of the rearranged JH/J kappa...
متن کاملDirect high‐throughput amplification and sequencing of immunoglobulin genes from single human B cells
Single cell Ig gene amplification and sequencing has been widely used for the molecular and functional assessment of human antibody repertoires and has led to the identification of recombinant monoclonal antibodies with therapeutic potential against diverse pathogens [1–3]. Due to the high reagent and Sanger sequencing costs, these antibody-cloning strategies are limited to the analysis of rela...
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The nested polymerase chain reaction (PCR) method was used for the amplification of the influenza A H1N1 virus hemagglutinin monoclonal antibody light-chain and heavy-chain genes. Sequence analysis of the obtained genes was then used to identify common cloning methods of the mouse immunoglobulin-kappa (Igκ) light-chain and heavy-chain variable gene regions. Twenty-two pairs of amplification pri...
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During B cell development, rearrangement and expression of Ig heavy chain (HC) genes promote development and expansion of pre-B cells accompanied by the onset of Ig light chain (LC) variable region gene assembly. To elucidate the signaling pathways that control these events, we have tested the ability of activated Ras expression to promote B cell differentiation to the stage of LC gene rearrang...
متن کاملAmplification and analysis of cDNA generated from a single cell by 5'-RACE: application to isolation of antibody heavy and light chain variable gene sequences from single B cells.
The technique of 5'-rapid amplification of cDNA ends (5'-RACE) is widely used to amplify unknown sequences at the 5' end of a messenger RNA (mRNA). However, conventional 5'-RACE is inappropriate for producing cDNAs from a single cell due to the small quantity of mRNA present in one cell. In this study, we report an improved 5'-RACE method that is suitable for generating cDNA from a single cell....
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ژورنال
عنوان ژورنال: Proceedings of the National Academy of Sciences
سال: 1992
ISSN: 0027-8424,1091-6490
DOI: 10.1073/pnas.89.16.7610