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Tcr repertoire diversity review9/6/2023 ![]() Thereby, we can amplify all TCR or BCR genes without bias.Īt the same time, we have developed our original, dedicated, fully automatic repertoire analysis program called Repertoire Genesis to process the data output from the NGS at high speed and accuracy. At Repertoire Genesis, Inc., we use the adaptor-ligation PCR method, which adds an adapter sequence at the 5′-end of the DNA, which is then subjected to PCR amplification using a pair of primer sets consisting of an adapter primer and a C region-specific primer. We also need to equally amplify all of the TCR genes, which are composed of a number of gene fragments. For this reason, we have utilized relatively long-read sequencers such as 454 sequencing (Roche) and MiSeq (Illumina). Next-generation sequencing (NGS) is possible for a large amount of sequencing, but the drawback is that length of determined nucleotide sequence is short. In the TCR repertoire analysis, to assign the V, D, J and C regions, a long-read sequence (400–600 bp) is required. consists of three technologies, namely long-read gene sequence, unbiased gene amplification technology, and dedicated bioinformatics software (Figure 2). The next-generation TCR repertoire analysis method of Repertoire Genesis, Inc. Three Technologies of The Next Generation TCR/BCR Repertoire Analysis In the precursor messenger RNA, splicing occurs in the VDJ region and C (constant) region, and it expresses as a functional TCR gene. In this process, insertion and/or deletion of nucleotides (N region) randomly occur between the V and D and D and J regions, and this increases the diversity of the gene. Subsequently, gene rearrangement occurs between the V and DJ genes. In the β chain gene, one of each of the D and J genes bond randomly. Gene rearrangement occurs during the differentiation and maturation of T cells. The TCR gene, as well as the immunoglobulin gene, consists of a number of gene fragments on the genome such as V (variable), D (diversity), J (joining), and C (constant) gene regions (Figure 1). This technology is very useful for obtaining antigen-specific TCR/BCR genes by analyzing samples from antigen stimulation, virus infection, and disease patients.Įfficient screening of complete human antibody genes Technical Details T Cell Receptor (TCR) Gene Structure It is possible to obtain antigen-specific TCR/BCR genes by determining nucleotide sequences of large number of TCR/BCR genes at the clonal level. The extent of immune diversity and its alterations may be used for developing a prognosis after transplantation. Although immune diversity is essential to host defense against infections by viruses and bacteria, it is not feasible to quantitatively evaluate immune diversity. TCR/BCR repertoire analysis is useful for evaluating the diversity of an immune system. In this manner, malignant cells present at a frequency of less than one in one hundred thousand (less than 0.001%) can be identified at a clonal level and thus, be useful for detection of minimal residual disease (MRD). Nucleotide sequences of TCR/BCR reads ranging from 10^5 to 10^7 can be determined by next-gen TCR/BCR repertoire analysis. But conventional methods such as flow cytometric analysis or immune histochemical staining are limited in sensitivity and accuracy. Because a single T cell or B cell proliferates abnormally in patients with certain kinds of leukemia and malignant lymphoma, detection of the malignant cells is used for diagnosing and estimating treatment effects. TCR/BCR repertoire analysis is useful for determining immunological status and abnormality. This collection of T/B lymphocytes characterized by TCR/BCR bearing different antigen specificities is known as the “TCR/BCR repertoire.” “Repertoire” is a French word describing a stock of skills or performances. The variety is estimated to reach 1018 for TCR and 1014 for BCR. They are activated to initiate an immune reaction in response to specific binding of their receptors to antigens such as tumors and viruses.Ī huge variety of TCRs and BCRs are created by the mechanism of gene rearrangement and somatic hypermutation to react to various antigens. The main types of lymphocytes, T cells and B cells, surface-express receptors that recognize antigens, T cell receptor (TCR) and B cell receptor (BCR), respectively. White blood cells composed of granulocytes, dendritic cells, macrophages, and lymphocytes, play an essential role in body defense by eliminating invading pathogens such as viruses and malignant cells. ![]()
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