Preparation method for comprehensively amplifying humanTCR beta chain library by adopting few degenerate primers

Abstract

The invention provides a preparation method for comprehensively amplifying a humanTCR beta chain library by adopting a small amount of degenerate primers, and relates to the technical field of amplification preparation of an immune repertoire sequencing library. Lymphocyte lysis and total RNA (Ribonucleic Acid) extraction: step one, preprocessing a sample, and then extracting RNA; step 2, reverse transcription: carrying out reverse transcription on the obtained total RNA of the lymphocyte to obtain cDNA (complementary deoxyribonucleic acid); step 3, multiplex PCR: carrying out a first round of PCR and a second round of PCR on the cDNA, and carrying out specific amplification on the TCR beta chain gene of the cDNA; 4, a sequencing linker sequence and index are added, the constructed library is short but contains a complete CDR3 segment, higher flux and lower price can be obtained, the adding amount of each specific primer and an amplification system are determined after a large number of experiments are optimized, the number of primer dimers can be greatly reduced, and the high-quality library is obtained.

Description

technical field [0001] The invention relates to the technical field of amplification and preparation of an immune repertoire sequencing library, in particular to a preparation method for comprehensively amplifying a humanTCRβ chain library by using a small amount of degenerate primers. Background technique [0002] The immune repertoire (Immune Repertoire, IR) refers to the sum of all functionally diverse B cells and T cells in the individual's circulatory system at any given time. The diversity of the immune system is crucial to the health of the body, and the more diverse it is , the healthier the body's immune system is, the stronger the ability to recognize and clear antigens. The body's acquired immunity can recognize countless antigen molecules and respond accordingly. The molecular basis is T cell surface receptors and B cells Surface receptors (TCR and BCR) specifically recognize antigen segments and activate immune responses. The T cell pool and B cell pool in the i...

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Problems solved by technology

[0003] The current NGS preparation technology for immune repertoire includes multiplex PCR and 5'RACE (Rapid Amplification of cDNA Ends). Failure will lead to wasted efforts. Even if the above reaction is smooth and smooth, some non-specific products or non-full-length products usually appear in the result, and the cost is high. In contrast, multiplex PCR library construction is simple, easy and low-cost. The advantages are reflected, and it has become the preferred method for building an immune group library. However, there is a drawback in multiplex PCR that primer dimers will form between different primers, and the more primer pairs, the more serious the dimer phenomenon, which greatly Reduced primer amplification efficiency

Moreover, the more pairs of primers, the higher the cost and difficulty of the experiment. At present, there are no more than two kinds of immune repertoire preparations at home and abroad. One is full primer amplification, and the V region gene of the variable region of TCR has 152, using the traditional multiplex PCR amplification technology route, requires hundreds of pairs of primers, which undoubtedly adds a lot of difficulty and cost to the amplification and preparation of the immune repertoire. The latter is local amplification, using a part of V region primers to amplify increase, using local detection of the whole, but this method is easy to cause deviations to the results, and the randomness is too strong, the repeatability is poor, and the experimental results are unstable. Therefore, the present invention uses a unique computer algorithm to design a V gene for 152 43 pairs of degenerate primers were optimized through a series of experimental conditions to achieve full coverage amplification of the humanTCRβ chain sequencing library with a small number of primers

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