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.

Active Publication Date: 2019-04-09
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 group and method for constructing human B cell immune repertoire with same on basis of high-throughput sequencing
  • Multiple primer group and method for constructing human B cell immune repertoire with same on basis of high-throughput sequencing
  • Multiple primer group and method for constructing human B cell immune repertoire with same on basis of high-throughput sequencing

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 group and a method for constructing a human B cell immune repertoire with the same on the basis of high-throughput sequencing and belongs to the field of molecular biological detection. The multiple primer group consists of a group of upstream primers and a group of downstream primers, and the upstream primers are formed by connecting a joint 1, a primer bar code 1, a sequencing primer 1 and specific primers V1-V18 designed for V regions (variable regions) in series; the downstream primers are formed by connecting a joint 2, a primer bar code 2, a sequencing primer 2 and specific primers Va, Vd, Ve, Vg and Vm designed for conserved sequences of gene C regions (constant regions) of IgA, IgD, IgE, IgG and IgM in series; sequences of the primer bar code 1 and the primer bar code 2 are different while the sequences of the sequencing primer 1 and the sequencing primer 2 are the same. By means of the method, a BCR (B cell receptor) immune repertoirecan be constructed on the basis of a DNA sample or an RNA sample, and diversity information of BCRs can be completely covered. 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 multiplex 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 an individual's circulatory system 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) that B cells recognize antigens and has antigen-binding specificity. BCR is composed of two heavy chains and two light chains. , the heavy chain is divided into variable region (V region), constant region (C region), transmembrane region and cytoplasmic region; light chain only has V region and C region. V region consists of VH and VL two domains , they are each composed of three complementarity determining regions (...

Claims

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Application Information

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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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