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BCR diversity detection kit and application thereof

A kit and a variety of technologies, applied in the field of molecular biology, can solve the problems of human BCR gene acquisition or analysis methods that need to be improved, and achieve the effect of improving the detection rate

Inactive Publication Date: 2017-06-13
GENEMIND BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the acquisition or analysis methods of human BCR genes still need to be improved

Method used

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  • BCR diversity detection kit and application thereof
  • BCR diversity detection kit and application thereof
  • BCR diversity detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1 of the present invention provides a method for preparing a B lymphocyte receptor (BCR) DNA sample, comprising the following steps:

[0070] (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. AS1114544 UK) to obtain relatively pure PBMCs;

[0071] (2) Use the PureLink Genomic DNA Mini Kit (Life Technology, Cat.No: K1820-00) kit to extract the genomic DNA of the cells obtained in step (1), and use Nanodrop2000 (Thermo) to measure the concentration and purity of the DNA, and then save the genomic DNA .

[0072] DNA extraction electrophoresis results such as figure 1 Shown in -a (genomic DNA fragments refer to lanes 1-2; M is DNA Marker).

Embodiment 2

[0074] Embodiment 2 of the present invention provides a method for constructing a high-throughput sequencing library of leukemia minimal residual lesion BCR using multiple PCR primers of the leukemia minimal residual lesion BCR library, comprising the following steps:

[0075] Using the genomic DNA obtained in Example 1 as the amplification template, take the BCR primers, and then use the MultiplexPCR kit from QIAGEN Company (article number: 206143), and configure a multiplex PCR system according to the kit instructions, wherein the BCR primers include upstream primers and downstream primers. The upstream primers are an upstream primer set consisting of nucleotide sequences shown in SEQ ID NO:1~SEQ ID NO:13, and the downstream primers are nucleotides shown in SEQ ID NO:14~SEQ ID NO:17 The downstream primer set composed of sequence.

[0076] Each upstream primer is mixed equimolarly, the total primer concentration is 10 micromolar, and each downstream primer is equimolarly mixe...

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Abstract

The invention provides a kit for detection BCR diversity. The kit comprises a set of multiple PCR primers. The multiple PCR primer contains upstream primers and downstream primers. The upstream primers are composed of a group of sequences which have a one-to-one correspondence with nucleotide sequences as shown in SEQ ID NO:1- SEQ ID NO: 13. Sequences in the upstream primer have 0-3 more or less nucleotides than the corresponding sequences in the SEQ ID NO:1- SEQ ID NO:13. The downstream primers are composed of a group of sequences which have a one-to-one correspondence with nucleotide sequences as shown in SEQ ID NO:14- SEQ ID NO:17. Sequences in the downstream primer have 0-3 more or less nucleotides than the corresponding sequences in the SEQ ID NO:14- SEQ ID NO:17. By the use of the kit, BCR sequences of human can be efficiently obtained, human specific BCR CDR3 sequences are obtained, and detection rate of low-copy-number B cell clones can be raised.

Description

[0001] related application [0002] This application is a divisional case of Chinese patent application 201510500391.8 with an application date of August 14, 2015, and titled "A Multiplex PCR Primer and Method for Constructing Leukemia Minimal Residual Lesion BCR Library Based on High-throughput Sequencing". technical field [0003] The invention belongs to the field of molecular biology and relates to a gene detection kit, in particular to a BCR diversity detection kit and its application. Background technique [0004] The antigen specificity of T / B lymphocytes is largely determined by the amino acid sequence of the T / B lymphocyte receptor CDR3 region. A large number of V and J gene fragments on the T / B cell locus will produce various rearrangements in the synthesis of receptors. Nucleotides between V-J, V-D and D-J junctions are inserted or deleted independently of the template, and high The frequency variation is similar, which further increases the potential diversity o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10C40B50/06
CPCC12Q1/6806C12Q1/6869C12Q1/6886C12Q2600/156C12Q2600/16C40B50/06C12Q2531/113C12Q2537/143C12Q2535/122C12N15/10C12N15/11C12Q1/68
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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