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Sequencing method for constructing variable region sequence library kit and variable region sequence

A variable region and kit technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of amplification bias, low amplification efficiency, easy loss of variable region sequence library sequence information, etc. question

Active Publication Date: 2021-11-09
GUANGZHOU HOMEY HEALTH TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In view of this, the present invention discloses a kit for constructing a variable region sequence library and a sequencing method for a variable region sequence, which can effectively solve the problems of low amplification efficiency and existence of amplification problems existing in the current variable region sequence library technology for constructing TCR or BCR. Bias and technical flaws that are prone to loss of sequence information in variable region sequence libraries

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  • Sequencing method for constructing variable region sequence library kit and variable region sequence
  • Sequencing method for constructing variable region sequence library kit and variable region sequence
  • Sequencing method for constructing variable region sequence library kit and variable region sequence

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Embodiment 1

[0063] The embodiment of the present invention provides a specific method for constructing a variable region sequence library, the steps of which are as follows:

[0064] One: template preparation

[0065] 1. Collect 5-10ml of human peripheral blood in an anticoagulant tube;

[0066] 2. Add the received fresh blood to three times the volume of RBC lysis buffer, and gently invert the centrifuge tube to mix evenly;

[0067] 3. Let stand at room temperature for 15 minutes;

[0068] 4. Centrifuge at 450xg for 10 minutes, carefully remove the supernatant, and leave the cell pellet;

[0069] 5. If the pellet is still obviously red, add RBC lysis buffer twice the volume of the original blood, and repeat steps 2 and 3;

[0070] 6. Suspend the pellet evenly with 600 μl PBS, take 200 μl for DNA extraction with TIANamp Genomics DNA kit;

[0071] 7. Primer preparation:

[0072] 7.1. Primers There are 52 TRBV, 13 TRBJ random, 14 IgHV, 6 IgHJ random, and 1 overhang primers, all of which ...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a kit for constructing a variable region sequence library and a sequencing method for the variable region sequence. The invention discloses a kit for constructing a variable region sequence library, comprising the following primers: a first primer group, a first adapter primer and a second primer group; the first primer group includes coding sequences that specifically recognize all subtypes of the J region The first primer; the first primer includes the nucleotide sequence of the coding sequence of the specific recognition J region connected in sequence, the proofreading random segment and the first adapter; the second primer group includes the specific recognition of the V region connected in sequence The second primers for the coding sequences of all subtypes; the second primers include a nucleotide sequence that specifically recognizes the coding sequences of the V region and a second linker. The invention is used to solve the technical defects of low amplification efficiency, bias in amplification and easy loss of sequence information of the variable region sequence library existing in the current variable region sequence library technology for constructing TCR or BCR.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for constructing a variable region sequence library and a sequencing method for the variable region sequence. Background technique [0002] "Immunity" is an extremely important self-defense function of the human body. Relying on its own immunity, it can resist a huge variety of diseases. Almost all diseases in the human body are closely related to immunity. The immune cells responsible for defending the body in the human microenvironment mainly include T cells, B cells, macrophages, dendritic cells, etc. These professional immune cells have unique structures and functions, and contain unique immune cell subsets and functional molecules. T cells and B cells are the main lymphocytes in the human body, which are responsible for cellular immunity and humoral immunity respectively. An in-depth understanding of the composition of T cells and B cells is helpful for the und...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6876C12Q1/6869
CPCC12Q1/6869C12Q1/6876C12Q2535/122C12Q2525/191
Inventor 许林浩黄智敏
Owner GUANGZHOU HOMEY HEALTH TECH
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