Construction method of T cell antigen receptor diversity sequencing library and kit

A technology for sequencing libraries and kits, used in chemical libraries, biochemical equipment and methods, and microbial determination/inspection, etc., can solve the problems of difficult primer design, uneconomical, and biased results.

Active Publication Date: 2018-03-09
CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In such prior art, firstly, the design of primers is very difficult; the competition between dozens of pairs of primers is inevitable, and it is easy to cause deviations in the results
In addition, after obtaining the TRB amplification products, it is necessary to add sequencing adapters to the amplified fragments and separately construct the sequencing library, which is cumbersome.
Moreover, from the perspective of cost control, the design of multiple pairs of primers and the addition of sequencing adapters to the amplified fragments are not economical.

Method used

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  • Construction method of T cell antigen receptor diversity sequencing library and kit
  • Construction method of T cell antigen receptor diversity sequencing library and kit
  • Construction method of T cell antigen receptor diversity sequencing library and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] 1. Experimental primers:

[0132] Table 1:

[0133]

[0134]

[0135] All the primers in Table 1 above were dissolved and made into a stock solution with a concentration of 100 μM. The working solution concentration is 10 μM.

[0136] 2. Experimental process:

[0137] 2.1 Total RNA extraction: total RNA was extracted from mouse spleen as a biological sample.

[0138] 2.2 Reverse transcription: TRBC-RT and 5TSA in the above table 1 are used as primers, using SuperScript TM II ReverseTranscriptase (Thermo Fisher#18064014) kit, perform primer-specific reverse transcription to generate cDNA template, the specific steps are as follows:

[0139] (1) RNA denaturation, incubation with primers:

[0140] Adjust the RNA concentration to 100-1000 ng / μL, take 2 μL, add 1 μL of TRBC-RT (10 μM), mix well, place in a 70°C water bath for 2 minutes, then place in a 42°C water bath for 2 minutes;

[0141] (2) Reverse transcription, through template replacement, introduce molec...

Embodiment 2

[0174] 1. Experimental primers:

[0175] Table 4:

[0176]

[0177]

[0178] All the primers in Table 1 above were dissolved and made into a stock solution with a concentration of 100 μM. The working solution concentration is 10 μM.

[0179] 2. Experimental process:

[0180] 2.1 Extraction of total RNA: 3 cases of human peripheral blood samples were used to separate white blood cells as biological samples, and total RNA was extracted.

[0181] 2.2 Reverse transcription: TRBC-RT and 5TSA in the above table 1 are used as primers, using SuperScript TM II ReverseTranscriptase (Thermo Fisher#18064014) kit, perform primer-specific reverse transcription to generate cDNA template, the specific steps are as follows:

[0182] (1) RNA denaturation, incubation with primers:

[0183] Take 700ng RNA, add 1 μL of TRBC-RT (10 μM), add water to 6.9 μl, mix well, and put in a PCR machine at 70°C for 2 minutes, and at 42°C for 2 minutes;

[0184] (2) Reverse transcription, through te...

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Abstract

The invention relates to a construction method of a T cell antigen receptor diversity sequencing library and a kit. The construction method provided by the invention comprises the following steps: amplifying regions V, D and J of a TRB gene expressed by a human or mouse T lymphocyte by utilizing a designed specific primer specific to a consensus sequence of a region C of the human or mouse TRB gene, synchronously introducing a next generation sequencing joint sequence into an amplified product, and directly sequencing the regions V, D and J of the TRB gene.

Description

technical field [0001] The invention relates to a method for constructing a T cell antigen receptor (TCR) sequencing library and a kit for constructing a T cell receptor sequencing library. Background technique [0002] Lymphocytes recognize specific antigens through their surface antigen recognition receptors, and the specificity of antigen recognition is reflected at the clonal level, that is, lymphocytes of the same clone have the same antigen receptors and recognize the same epitope. T cell antigen receptor (TCR) is a molecular structure on the surface of T cells that specifically recognizes antigens and binds antigen peptide-MHC molecules. [0003] TCR can be divided into two types: TCRα / β and TCRγ / δ. Peripheral blood T cells are mainly TCRα / βT cells, which are the main cells that mediate the body's specific cellular immune response. The TCR diversity of peripheral blood T cells is determined by the V regions of the α chain and the β chain. Vα and Vβ each have three hy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12N15/10C40B50/06
CPCC12N15/1096C12Q1/6806C40B50/06C12Q2531/113C12Q2525/191C12Q2549/119
Inventor 冯林张雅静郭丽萍刘玲玲匡满超程书钧张开泰肖汀张文
Owner CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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