Multi-PCR primer and method for constructing leukemia minimal residual disease TCR library based on high-throughput sequencing

A minimal residue and leukemia technology, applied in the field of molecular biology, can solve the problems of labor-intensive, unfavorable clinical standardized detection, interference of mpFC detection, etc., and achieve the effect of improving the detection rate

Active Publication Date: 2015-12-16
GENEMIND BIOSCIENCES CO LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Although the detection sensitivity of mpFC for recurrent disease is 10 -4 , but the complex multi-dimensional data depends on the analysis of the experimenter, and the influence of human factors is large, which is not conducive to clinical standardized detection
In addition, the expression levels of leukemia antigens interfered with the detection of mpFC in MRD after chemotherapy
Relying on molecular means can improve the sensitivity of detecting MRD, which can reach 10 -5 However, real-time quantitative PCR needs to design special primers according to patients to amplify the diverse rearranged sequences, which is expensive, labor-intensive, and difficult to form a standardized experimental process

Method used

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  • Multi-PCR primer and method for constructing leukemia minimal residual disease TCR library based on high-throughput sequencing
  • Multi-PCR primer and method for constructing leukemia minimal residual disease TCR library based on high-throughput sequencing
  • Multi-PCR primer and method for constructing leukemia minimal residual disease TCR library based on high-throughput sequencing

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Embodiment 1

[0056] Embodiment 1 of the present invention provides a method for preparing a T lymphocyte receptor (TCR) DNA sample, comprising the following steps:

[0057] (1) Collect 10 milliliters (ml) of fresh peripheral blood samples each, and operate according to the instructions of the LymphoPrep kit (Axis-shield, Cat. No. AS1114544UK) to obtain relatively pure PBMCs;

[0058] (2) Use the PureLink GenomicDNA MiniKit (LifeTechnology, Cat. No: K1820-00) kit to extract the genomic DNA of the cells obtained in step (1), measure the concentration and purity of the DNA with Nanodrop2000 (Thermo), and then save the genomic DNA.

[0059] DNA extraction electrophoresis results such as figure 1 -a (see lanes 1-2 for genomic DNA fragments; M is DNAMarker).

Embodiment 2

[0061] Embodiment 2 of the present invention provides a method for constructing a TCR high-throughput sequencing library of minimal residual lesion lesion by using multiple PCR primers of the minimal residual lesion TCR library of leukemia, including the following steps:

[0062] Using the genomic DNA obtained in Example 1 as an amplification template, take TCR primers, and then use QIAGEN’s MultiplexPCR kit (article number: 206143) to configure a multiplex PCR system according to the kit instructions, wherein the TCR primers include upstream primers and downstream primers. The upstream primers are an upstream primer set composed of nucleotide sequences shown in SEQIDNO:1-SEQIDNO:25, and the downstream primers are a downstream primer set composed of nucleotide sequences shown in SEQIDNO:26-SEQIDNO:38.

[0063] Each upstream primer is mixed equimolarly, the total primer concentration is 10 micromolar, and each downstream primer is equimolarly mixed, the total primer concentratio...

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Abstract

The invention provides a multi-PCR primer for constructing a leukemia minimal residual disease TCR library based on high-throughput sequencing; the primer comprises a forward primer and a reverse primer, wherein the forward primer is a forward primer group consisting of nucleotide sequences which are 0-3 bases more than or less than nucleotide sequences as shown in SEQ ID NO: 1-SEQ ID NO: 25; and the reverse primer consists of nucleotide sequences which are 0-3 bases more than or less than nucleotide sequences as shown in SEQ ID NO: 26-SEQ ID NO: 28. The multi-PCR primer group provided by the invention can be used for effectively constructing a T lymphocyte receptor high-throughput sequencing library of patients of leukemia, and abundant leukemia minimal residual disease TCR rearrangement information can be obtained.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a multiplex PCR primer and a method for constructing a TCR library of leukemia minimal residual lesions based on high-throughput sequencing. Background technique [0002] Leukemia is a malignant clonal disease of the blood system characterized by an increase in immature cells in the bone marrow and / or peripheral blood. At the time of first diagnosis, the total number of leukemia cells in the patient's body was about 10 12 , after chemotherapy to achieve morphological complete remission (Complete Remission, CR), the total number of residual leukemia cells is less than 10 9 , this morphological method is difficult to detect, and a small amount of leukemia cells still remain in the body is called minimal residual disease (minimal residual disease, MRD). Residual MRD in the body causes >50% relapse rate in patients with acute T-lymphoblastic leukemia. Therefore, reg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/68C40B50/06
CPCC12N15/10C12N15/11C12Q1/68C40B50/06
Inventor 葛良进刘松林群婷刘丽春曾立董黄莎莎黄亮李改玲
Owner GENEMIND BIOSCIENCES CO LTD
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