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A kind of design method of primer set and its application

A design method and primer set technology, applied in the biological field, can solve the problems of low coverage, shortened target gene fragments, and decreased amplification efficiency, and achieve the effects of improving reaction efficiency, reducing mismatch rate, and improving coverage.

Active Publication Date: 2022-05-17
SUZHOU HONGXUN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method requires very long primers, which will lead to a decrease in amplification efficiency and shortened target gene fragments, and requires extremely high throughput to cover all UIDs. This method is currently mainly used in RNA sequencing of IGH / TCR
[0007] For the primer design of the immune repertoire, it is difficult to design an upstream primer to identify the 5' end sequence of the BCR gene in the current technology. Primer design based on the FR1 region will cause the upstream information of the antibody to not be covered, making the sequenced gene information incomplete ; For the primers in literature 2 (Wang X, Stollar BD. Human immunoglobulin variable regiongene analysis by single cell RT-PCR. J Immunol Methods 2000; 244:217–25), although the number of primers is small, the coverage is low
The immune library sequencing method of multiple primer PCR technology requires dozens of pairs of primers only for BCR sequencing. In this way, the huge number of primers will make the amplification efficiency and specificity of the entire PCR poor.

Method used

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  • A kind of design method of primer set and its application
  • A kind of design method of primer set and its application
  • A kind of design method of primer set and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Design of primer set for human B cell immune repertoire

[0068] (1) Extract human germline gene

[0069] Download the human heavy chain germline gene from the IMGT database (http: / / imgt.org / vquest / refseqh.html): 361 genes were extracted, and the sequence information of 1 FASTA is as follows:

[0070] >M99641|IGHV1-18*01|Homo sapiens|F|V-REGION|188..483|296nt|1|||||296+24=320|||

[0071] (2) Primer selection region positioning

[0072] Align 361 sequences to chromosome 14 "hs_ref_GRCh38.p12_chr14.fa", and extract the base sequence group 150bp before the start site of the sequence;

[0073] (3) Candidate primer library design

[0074] According to each 20bp, 69430 primers were formed as the starting data set to form the candidate primer library.

[0075] (4) cluster clustering analysis

[0076] Classify the candidate primer library with Cd-hit software, set the -c parameter to 0.9, and perform cluster analysis; sort the clusters according to size, select t...

Embodiment 2

[0103] Example 2 Primer set performance evaluation

[0104] (1) Sample preparation

[0105] Use human lymphocyte separation medium to separate peripheral blood B lymphocytes, extract RNA, and reverse transcribe cDNA. The steps are as follows:

[0106] 1) 10 milliliters (mL) of fresh peripheral blood samples (5) were collected, and operated according to the instructions of the LymphoPrep kit to obtain relatively pure peripheral blood mononuclear cells (PBMC);

[0107] 2) Extract RNA, and use Nanodrop2000 to measure the concentration and purity of RNA, use TIANScriptM-MLV Reverse Transcriptase Kit (Product No. ER104), and obtain cDNA after reverse transcription (i.e. samples: ST-1, ST-2, ST-3, ST -4, ST-5) are amplification templates, standby;

[0108] The reverse transcription reaction steps are as follows:

[0109] a) System configuration:

[0110] Configure the reverse transcription reaction system according to Table 2.

[0111] Table 2

[0112] components D...

Embodiment 3

[0144] Example 3 Analysis of biological information of the immune repertoire

[0145] Using the designed primer set (SEQ ID NO: 1-SEQ ID NO: 16) as the upstream primer, and SEQ ID NO: 17 as the downstream primer, the amplified human B cell immune repertoire was used for subsequent bioinformatics analysis. The result is as follows:

[0146] (1) CDR3 region length analysis

[0147] In the CDR3 amino acid sequence, a change of one amino acid (aa) may lead to a change in the conformation of the receptor. Therefore, the change in the length of the CDR3 amino acid sequence can reflect the diversity of the CDR3 gene junction region. The sequence information of the immune repertoire is analyzed by Igblast software Finally, the CDR3 length distribution of the immune repertoire (see figure (2)), and the use of germline genes (germline) in the CDR3 region (see figure (3)) were obtained.

[0148] Depend on figure 2 It can be found that the amino acid length of CDR3 is mainly concentra...

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Abstract

The invention provides a design method and application of a primer set. According to the needs of amplification length, the sequence before the FR1 start site of the antibody is subtly extracted from the upstream of the FR1 start site of the antibody, and the sequence is extracted from the 5' end of the primer set. Starting from the first base, the fixed-length primer sequences are sequentially shifted and cut to form a candidate primer library. After cluster analysis, the primer set of the immune group library is screened. The primer set obtained by this method can significantly improve the immune group of the multiplex PCR method. The coverage and experimental efficiency of library amplification, reducing the amplification mismatch rate, the method is simple, and the cost is saved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a design method and application of a primer set, in particular to a design method and application of a primer set of an immune repertoire. Background technique [0002] Immune repertoire (Immune Repertoire, IR) refers to the sum of polymorphisms of all functionally diverse B cells and T cells in the circulation of an individual at any given time. The immune cells responsible for defending the body mainly include T cells, B cells, macrophages, and dendritic cells. T and B cells are the main lymphocytes in the human body, responsible for cellular immunity and humoral immunity respectively, among which B cells account for about 20% of the total number of peripheral lymphocytes. BCR is composed of two heavy chains and two light chains connected, in which the heavy chain is divided into variable region (V region), constant region (C region), transmembrane region and cytoplasmic region; the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G16B5/00G16B25/10G06K9/62
CPCG06F18/23
Inventor 吴婷婷李彦敏陈淑美蔡晓辉杨平
Owner SUZHOU HONGXUN BIOTECH CO LTD
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