Multiple primer group and method for constructing human B cell immune repertoire with same on basis of high-throughput sequencing
An immunological library and primer set technology, which can be used in biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, etc., and can solve problems such as complex operations.
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Embodiment 1
[0104] A multiplex primer set, consisting of a set of upstream primers and a set of downstream primers, the upstream primers are formed in series by adapter 1, primer barcode 1, sequencing primer 1, and specific primers V1-V18 designed for the V region of the variable region; The downstream primers are composed of adapter 2, primer barcode 2, sequencing primer 2, and specific primers Va, Vd, Ve, Vg, and Vm designed for the conserved sequences of the gene constant regions C of IgA, IgD, IgE, IgG, and IgM in series. The sequences of primer barcode 1 and primer barcode 2 are different;
[0105] Linker 1 sequence is AAT GAT ACG GCG ACC ACC GAG ATC TAC AC;
[0106] Linker 2 sequence is CAA GCA GAA GAC GGC ATA CGA GAT.
[0107] The sequences of sequencing primer 1 and sequencing primer 2 are both GCT CTT CCG ATC T.
[0108] Primer barcode 1 and primer barcode 2 are represented by NNNNNN (NN), consisting of 6 or 8 nucleotides, which is convenient for different sample libraries to b...
Embodiment 2
[0123] Use DNA samples to amplify the target sequence by multiplex PCR for library construction:
[0124] 1. Rapid isolation of peripheral blood lymphocytes
[0125] 1) Inspection: Take out the lymphocyte anticoagulant separation tube and observe whether there is free separation liquid on the separation gel. If there is, centrifuge at 2000g for 1 min at room temperature.
[0126] 2) Sampling: Add 5ml of peripheral blood to the anticoagulant separation tube.
[0127] 3) Centrifugation: 800g, soft centrifugation at room temperature for 15min.
[0128] 4) Discard the plasma layer with a Pasteur pipette or a pipette until it is close to the PBMC cell layer, then carefully suck out the PBMC (buffy coat layer: between the separation gel and plasma), and transfer to a new 15ml centrifuge tube.
[0129] 5) Add physiological saline or 1*PBS to 15ml, wash once, centrifuge at 300g at soft room temperature for 10min.
[0130] 6) Discard the supernatant, add 2ml normal saline or 1*PBS, ...
Embodiment 3
[0170] When using RNA samples, the target sequence is amplified by multiplex RT-PCR for library construction:
[0171] 1. Rapid isolation of peripheral blood lymphocytes
[0172] 1) Inspection: Take out the lymphocyte anticoagulant separation tube and observe whether there is free separation liquid on the separation gel. If there is, centrifuge at 2000g for 1 min at room temperature.
[0173] 2) Sampling: Add 5ml of peripheral blood to the anticoagulant separation tube.
[0174] 3) Centrifugation: 800g, soft centrifugation at room temperature for 15min.
[0175] 4) Discard the plasma layer with a Pasteur pipette or a pipette until it is close to the PBMC cell layer, then carefully suck out the PBMC (buffy coat layer: between the separation gel and plasma), and transfer to a new 15ml centrifuge tube.
[0176] 5) Add physiological saline or 1*PBS to 15ml, wash once, centrifuge at 300g at soft room temperature for 10min.
[0177] 6) Discard the supernatant, add 2ml normal sali...
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