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Standard for DNA homologous recombination repair gene detection and preparation method thereof

A gene detection and homologous recombination technology, applied in the field of biomedicine, can solve problems such as the inability to use plasmid standard products, gene abnormalities, and large differences

Pending Publication Date: 2021-04-27
南京科佰基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There are many detection kits and detection methods developed around HRR-related genes, such as Q-PCR, AMRAS-PCR, NGS, etc. These methods can make qualitative and quantitative judgments on HRR-related genes in principle, and then can analyze clinical Significance, but for the quality control of any method, the performance evaluation of the method, how about the detection limit? How about repeatability? How about accuracy? There is no ideal standard product on the market to answer these questions
[0007] The main reason is that there are many genes related to HRR, and there are no hot spot mutations in each gene. That is to say, any Pathogenic, Likely Pathogenic, or even Uncertain Significance may cause gene abnormalities, which in turn need to be detected. concern, need to be detected
If you follow this logic, the practice of plasmid standard products in the past is definitely inappropriate. First, the plasmid is a pure product, not a whole genome, which is very different from clinical samples. Second, it is impossible to design all the hotspot mutations of so many genes you want. In the IVD review guidelines for Class III medical devices, it is also clearly recommended that plasmid standards cannot be used

Method used

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  • Standard for DNA homologous recombination repair gene detection and preparation method thereof
  • Standard for DNA homologous recombination repair gene detection and preparation method thereof
  • Standard for DNA homologous recombination repair gene detection and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Select 60 strains of tumor cells, as shown in Table 1, culture 60 kinds of cells according to the instructions of the cells, digest the cells with 0.25% trypsin (Gibco#25200056), count them with countess II (thermo AMQAX1000), and collect each cell 1×10 6 cell suspension;

[0038] Table 1

[0039]

[0040]

[0041]

[0042] 2. Use the Cell Genomic DNA Extraction Kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to extract the genomic DNA of 60 kinds of cells respectively (see the manufacturer's instructions for specific operations);

[0043] 3. Perform QC detection on the extracted gDNA:

[0044] 3.1 The prepared DNA was quantified using qubit 3.0;

[0045] 3.1.1 The sample to be tested and Qubit TM Take the dsDNA HS Assay kit out of the refrigerator and let it cool to room temperature;

[0046] 3.1.2 Configure the dye (Buffer: dye = 200: 1);

[0047] 3.1.3 Add 18ul Qubit to the 1.5ml centrifuge tube TM dsDNA HS Buffer and 2ul sample to be test...

Embodiment 2

[0106] This embodiment is a standard product for DNA homologous recombination repair gene detection, the standard product includes a gene panel;

[0107] The gene panel includes 28 genes, and the corresponding names of the 28 genes are as follows:

[0108] BRCA1, BRCA2, ATM, ATR, BARD1, BLM, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCC, FANCD2, FANCF, FANCI, FANCL, FANCM, MRE11A, NBN, PALB2, PPP2R2A, RAD50, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, and RPA1;

[0109] The above gene panels are derived from HCC1143, HCC1599 and HCC38 cell lines, and the mass ratio of gene DNA derived from HCC1143, HCC1599 and HCC38 cell lines is 2:3:4.

Embodiment 3

[0111] The present embodiment is the preparation method of embodiment 2 standard substance, and this preparation method comprises the steps:

[0112] (a) Obtain HCC1143, HCC1599 and HCC38 cell lines, culture three kinds of cells according to the instructions of the cells, use 0.25% trypsin (Gibco#25200056) to digest the cells, use countess II (thermo AMQAX1000) to count, and collect 1×10 cells per cell 6 cell suspension;

[0113] (b) Genomic DNA in HCC1143, HCC1599 and HCC38 cell lines were extracted using a cell genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.);

[0114] (c) Genomic DNA is detected according to the QC detection method in Example 1;

[0115] (d) After passing the test, use the Bioruptor ultrasonic interrupter to randomly interrupt the genomic DNA to 350bp, perform end repair and add A tails on the interrupted DNA fragments, and then perform the first PCR amplification, and then use IDT The xGen ExomeResearch Panel v2 kit capture...

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Abstract

The invention discloses a standard substance for DNA homologous recombination repair gene detection and a preparation method and application thereof. The standard comprises a gene panel; the gene panel includes 28 types of genes, and corresponding names of the 28 types of genes are as follows: BRCA1, BRCA2, ATM, ATR, BARD1, BLM, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCC, FANCD2, FANCF, FANCI, FANCL, FANCM, MRE11A, NBN, PALB2, PPP2R2A, RAD50, RAD51B, RAD51C, RAD51D, RAD52, RAD54L and RPA1. According to the standard, by performing next generation sequencing (NGS) detection on specific tumor cell strains, 28 types of genes related to HRR are screened out, and through mixing according to a specific ratio, each gene contains various mutation types and mutation frequency full coverage, then the standard is formed after accurate quantification, and then the standard can be used for performance evaluation in HRR detection.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a standard product for DNA homologous recombination repair gene detection and a preparation method thereof. Background technique [0002] Breast cancer (BC) has become the most common malignant tumor among Chinese women, and its incidence is increasing year by year. With the progress of genome-wide association studies (GWAS), a series of DNA damage repair-related gene mutations and single nucleotide polymorphisms (single nucleotide polymorphisms, SNP) sites that are closely related to the occurrence and development of BC have been discovered . The tumor suppressor network with BRCA1 / 2 as the core is mainly involved in the repair process of DNA double strand breaks (doublestrands breaks, DSB) caused by various factors including ionizing radiation, and regulates homologous recombination repair by forming functional complexes with various proteins (homologous recombination rep...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6806C40B50/06
CPCC12Q1/6886C12Q1/6806C40B50/06C12Q2600/156C12Q2535/122C12Q2531/113
Inventor 蒋涛华傅坚刚邵悦
Owner 南京科佰基因科技有限公司
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