Variable region sequence library construction kit and sequencing method of variable region sequence

A variable region and kit technology, applied in the biological field, can solve the problems of easy loss of variable region sequence library sequence information, low amplification efficiency, and bias in amplification.

Active Publication Date: 2018-10-26
GUANGZHOU HOMEY HEALTH TECH
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  • 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...

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  • Variable region sequence library construction kit and sequencing method of variable region sequence
  • Variable region sequence library construction kit and sequencing method of variable region sequence
  • Variable region sequence library construction kit and sequencing method of 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 technical field of biology, and particularly relates to a variable region sequence library construction kit and a sequencing method of a variable region sequence. The variable region sequence library construction kit comprises a first primer group, primers of first connectors and a second primer group, the first primer group comprises first primers which specifically recognize all subtype coding sequences in a J region, each first primer comprises a nucleotide sequence, a proofreading random section and the first connector which are connected successively, the nucleotide sequences specifically recognize the coding sequences in the J region, the second primer group comprises successively connected second primers which specifically recognize all subtype coding sequences in a V region, and each second primer comprises a nucleotide sequence which specifically recognizes the coding sequences in the V region and a second connector. The variable region sequence library construction kit and the sequencing method of the variable region sequence are used for overcoming the technical defect that an existing TCR (T cell receptor) or BCR (B cell receptor) variable region sequence library construction technology is low in amplification efficiency, biased in amplification and prone to losing sequence information of a variable region sequence library.

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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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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