Method for detecting rearrangement clonality of correlative genes of lymphocyte

A clonality and gene technology, applied in the field of clonal detection of lymphocyte-related gene rearrangements, can solve the problems of increasing detection costs, inability to detect somatic hypermutation at the same time, and inability to analyze rearrangements in depth.

Active Publication Date: 2018-05-08
北京旌准医疗科技有限公司
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  • Abstract
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Problems solved by technology

However, there are still big problems in the clinical application of this method of detection: 1. There are many gene sites to be detected, and one sample needs multiple PCR reactions for detection, which increases the detection cost and complicates the operation process; 2. It cannot be performed More detailed and in-depth pedigree analysis can only detect monoclonal rearrangements for IG or TCR genes, but cannot in-depth analysis of which family gene in the V region or J region is involved in the rearrangement, providing more detailed information for the clinic; 3. Detection limit There are still major limitations. The current PCR-capillary electrophoresis method can only detect 1% of monoclonal rearrangements, which cannot meet the needs of clinical MRD detection; 4. Can not detect somatic hypermutation at the same time

Method used

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  • Method for detecting rearrangement clonality of correlative genes of lymphocyte
  • Method for detecting rearrangement clonality of correlative genes of lymphocyte
  • Method for detecting rearrangement clonality of correlative genes of lymphocyte

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Experimental program
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Effect test

Embodiment 1

[0071] The specific operation process that the present invention takes in order to realize the above object is:

[0072] (1) Selection and verification of target fragment-specific primers:

[0073] The BIOMED-2 gene rearrangement primer detection system has been proved to have good specificity and sensitivity through a large number of clinical experiments. The gold standard for gene clonality rearrangement. However, with the development of next-generation sequencing, there have been no reports of applying such high-specificity and high-sensitivity primer combinations to the detection of next-generation sequencing. Therefore, BIOMED-2 classic primers (such as JJM vanDongen, Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspectedlymphoproliferations: Report of the BIOMED-2 Concerted Action BMH4-CT98-3936, Leukemia (2003) 17, 2257–2317) as the specific amplification primers of this method...

Embodiment 2

[0097] 1. Materials

[0098] 1.1 Sample selection:

[0099] 3 cases of nucleic acid were extracted from paraffin sections of clinical samples of unknown type; 1 case of nucleic acid extracted from ATCC CRL-2959 cell line was used as a positive control; 1 case of clinically excised tonsil tissue frozen section was used as a negative control; 1 case of pure water was used as a blank control

[0100] 1.2 Detection of genes:

[0101] Choose to perform clonality detection on IGH gene rearrangement, and use primers that match the sequence of the target region of the sample as shown in the table below:

[0102] Table 2

[0103]

[0104] Choose to perform clonality detection on TCRG gene rearrangement, and use primers to match the sequence of the target region of the sample as shown in the table below:

[0105] table 3

[0106]

[0107]

[0108] 1.3 PCR reagents

[0109] Including AntiTaq enzyme (Roche) and Buffer for amplification, dNTP and MgCl 2 the solution

[0110...

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Abstract

The invention discloses a method for detecting rearrangement clonality of correlative genes of lymphocyte and belongs to the technical field of biology. According to the method, a high-throughput next-generation sequencing method is adopted for detecting rearrangement clonality of the correlative genes of lymphocyte tissue, and specifically, a multifunctional primer and a PCR reagent are utilizedto conduct multiplication on samples to be detected at first to obtain a multi-sample gene locus multiplication sublibrary, wherein both ends of the multiplication sublibrary are connected with DNA segments of different connectors, one side of the multiplication sublibrary is a sequencing connector which can contain a sample tag for distinguishing sequencing results of the different samples, and the other side of the multiplication sublibrary is a fixed connector used for connecting caught particles; then, high-throughput sequencing is conducted on the multiplication sublibrary, sequencing results are subjected to classified sorting according to a tag sequence, the samples and target genetic loci, and whether or not there is clonality rearrangement of target genes is analyzed. The method has the advantages that the detection throughput, the sensitivity and the specificity are high, the sensitivity can reach 0.01%, and the DNA concentration of the samples can be lowered to 0.1-1ng/microliter.

Description

technical field [0001] The invention relates to a method for detecting the clonality of lymphocyte-related gene rearrangement, and belongs to the field of biotechnology. Background technique [0002] Lymphocytes are produced by lymphoid organs and are a type of cell line with the body's immune recognition response function, which can be divided into B cells, T cells and NK cells. There are receptor proteins on the surface of lymphocytes that can participate in immune reactions, and can specifically bind to antigens or antibodies, such as immunoglobulin (IG) on the surface of B lymphocytes and T cell receptor (TCR) on the surface of T lymphocytes. Taking the IGH gene expressing immunoglobulin heavy chain polypeptide as an example, in undifferentiated lymphocytes, the IGH gene is mainly composed of a variable region (variabl V), a diversity region (diversity D), and a joining region (joining J). Each gene region is composed of multiple gene segments. With the differentiation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2525/191C12Q2535/122C12Q2531/113
Inventor 袁太明刘明坤叶锋
Owner 北京旌准医疗科技有限公司
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