A kind of design method of primer set and its application

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...

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.

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