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Multiple primer set and method for constructing human b-cell immune repertoire based on high-throughput sequencing using the primer set

A primer set and multiplex technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as complex operations, improve construction efficiency, reduce cumbersome library construction steps, and save time Effect

Active Publication Date: 2021-11-26
SHANDONG ACV BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the preparation of traditional sequencing libraries requires procedures such as sample nucleic acid extraction, enzyme pretreatment or mechanical shearing, adding adapters for ligation, PCR amplification, etc., and the operation is complicated.

Method used

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  • Multiple primer set and method for constructing human b-cell immune repertoire based on high-throughput sequencing using the primer set
  • Multiple primer set and method for constructing human b-cell immune repertoire based on high-throughput sequencing using the primer set
  • Multiple primer set and method for constructing human b-cell immune repertoire based on high-throughput sequencing using the primer set

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses a multiple primer set and a method for using the primer set to construct a human B cell immune repertoire based on high-throughput sequencing, belonging to the field of molecular biology detection. The multiplex primer set of the present invention is composed of a set of upstream primers and a set of downstream primers. The upstream primers are connected 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 formed in tandem by adapter 2, primer barcode 2, sequencing primer 2, specific primers Va, Vd, Ve, Vg, Vm designed for the conserved sequence of the gene constant region C region of IgA, IgD, IgE, IgG, IgM ; The sequences of primer barcode 1 and primer barcode 2 are different; the sequences of sequencing primer 1 and sequencing primer 2 are the same. By using the method of the present invention, a BCR immune repertoire can be constructed based on DNA samples or RNA samples, which can fully cover the diversity information of BCR. Moreover, the construction efficiency is high and the cost is low.

Description

technical field [0001] The invention relates to the field of molecular biology detection, in particular to a multiple primer set and a method for constructing a human B cell immune repertoire based on high-throughput sequencing using the primer set. Background technique [0002] The immune repertoire is the sum of all functionally diverse B and T cells in the circulation of an individual at any given time. [0003] Human lymphocytes mainly include T cells and B cells. B cell antigen receptor ((B cell receptor, BCR) is a membrane surface immunoglobulin (SmIg) for B cells to recognize antigens, and has antigen binding specificity. BCR is composed of two heavy chains and two light chains connected , where the heavy chain is divided into variable region (V region), constant region (C region), transmembrane region and cytoplasmic region; the light chain has only V region and C region. The V region consists of two domains, VH and VL , each of which is composed of three complemen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C40B50/06C12Q1/6869
CPCC12Q1/6869C40B50/06C12Q2535/122C12Q2537/143C12Q2537/165
Inventor 李艳艳邱盟轩靖相密汪朝晖
Owner SHANDONG ACV BIOTECH CO LTD
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