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Quality control method and kit for detecting human brca1/2 gene variation based on high-throughput sequencing

A technology for detecting kits and gene mutations, which is applied in biochemical equipment and methods, and microbial determination/inspection, etc. It can solve the problems of no obvious site specificity, low detection efficiency, and high cost, and reduce detection quality control Cost, efficiency improvement effect

Active Publication Date: 2020-02-18
上海思路迪医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing detection of fetal chromosomal aneuploidy (T21, T18, T13) uses semiconductor sequencing method. Due to the different purposes of analysis, such detection analyzes the variation at the chromosome level, so it cannot be used to target gene variation as Purpose Detection Analysis
Other gene mutation detection products, such as EGFR hotspot mutation detection, use PCR-fluorescent probe method, fluorescent PCR-capillary electrophoresis method, flow fluorescence hybridization method, FISH, sequencing method, Taqman-ARMS method, etc., because they can only Detecting a mutation at a specific site has low detection efficiency and high cost, and there is no specific hotspot for the mutation of the BRCA1 / 2 gene, and the incidence of mutation has no obvious site specificity, so conventional methods are not suitable for this type of mutation However, it cannot meet the needs of BRCA1 / 2 gene detection, which requires detection and analysis of the entire coding region of the target gene.

Method used

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  • Quality control method and kit for detecting human brca1/2 gene variation based on high-throughput sequencing
  • Quality control method and kit for detecting human brca1/2 gene variation based on high-throughput sequencing
  • Quality control method and kit for detecting human brca1/2 gene variation based on high-throughput sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Preparation of quality control products for human BRCA1 / 2 gene mutation detection

[0036] 1.1. Purchase from ATCC to obtain four commonly used stable passage human tumor cell lines HCC1599, HCT-15, Jurkat, NCI-H720.

[0037] 1.2. The four human tumor cell lines were cultured with a special medium, and the culture conditions: 37°C constant temperature, 5% CO 2 , Humidity 50%. Cultivate until the cell density reaches 80-90% of the culture dish area and pass it down. Collect in the logarithmic phase of cell growth, centrifuge at 800-1000r / min to collect the precipitate, extract the genomic DNA of each cell line, and purify it by column. Eluted.

[0038] 1.3. Dilute the purified genomic DNA of each cell line to 100±5ng / μL with Tris-EDTA buffer solution. The appearance is a transparent liquid without visible impurities, and the purity is 1.9>OD260 / 280>1.7. Obtain the quality control material DNA.

[0039] 1.4. Use Sanger sequencing method to sequence the genomic DNA of...

Embodiment 2

[0041] Example 2: Preparation of human BRCA1 / 2 gene mutation detection kit

[0042] 2.1. Design and synthesize multiple capture probes for different target regions on the human BRCA1 / 2 gene. The collection of all capture probes can cover all the coding exon regions and exon-intron junctions of the human BRCA1 / 2 gene Region; the capture probe has a biotin label; the sequence of the capture probe is shown in Table 1.

[0043] Table 1 Human BRCA1 / 2 gene mutation detection hybridization capture probe sequence

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[0073] 2.2. Mix all the above capture probes with the same mass, and dilute the mixture to a working concentration of 1.5PM (PM = picomoles / liter) and store at -20°C.

[0074] 2.3. Separate the capture probe mixture and the q...

Embodiment 3

[0077] Example 3: Sequencing detection of human BRCA1 / 2 gene mutation

[0078] The instrument used for sequencing in this example is NextSeq500.

[0079] The preparation method of the positive quality control is the same as in Example 1.

[0080] 3.1. Extract human genomic DNA from 20 positive blood samples, and use the positive quality control products directly without extraction.

[0081] 3.2. Fragmentation of DNA samples and quality control products

[0082] Take 200ng for each human genomic DNA sample, and 200ng for quality control products. If it is less than 50μL, use PCR-gradewater to make up to 50μL, shake and mix well, and centrifuge briefly. Then transfer all the solution to a microtube, and select an interrupt program with a fragment length of 200-300bp to fragment the genomic DNA sample and quality control DNA. Take 5μL of the fragmented sample for agarose gel electrophoresis.

[0083] 3.3. Library construction

[0084] 3.3.1. End repair and joint connection

[0085] 0.2ml PC...

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Abstract

The invention relates to a quality control method and kit for detecting human BRCA1 / 2 gene variation based on high-throughput sequencing, and their application. The present invention extracts the genomic DNA of a plurality of BRCA1 / 2 gene mutation-positive human tumor cell lines, and uses the Sanger sequencing method to determine the mutation-positive sites as positive control sites, and wild-type sites as negative control sites. These genomic DNAs Mix in a certain ratio to obtain a quality control product that can be used for high-throughput sequencing to detect human BRCA1 / 2 gene variation. The kit of the present invention comprises a human BRCA1 / 2 gene variation detection quality control product.

Description

[0001] Invention field [0002] The invention relates to the field of gene detection, in particular to a quality control method and kit for detecting human BRCA1 / 2 gene mutation based on high-throughput sequencing. Background technique [0003] Breast cancer is one of the most common malignant tumors that seriously affect women's health and even endanger their lives. According to statistics, its incidence accounts for 7-10% of various malignant tumors throughout the body. The incidence of breast cancer is often related to genetics. In addition, the incidence of breast cancer is higher in women between 40-60 years of age and before and after menopause. [0004] Breast cancer can be divided into two categories: sporadic and hereditary. Among them, hereditary breast cancer accounts for about 5%-10% of the incidence of breast cancer. Breast cancer susceptibility genes BRCA1 (the first familial breast and ovarian cancer susceptibility gene) and BRCA2 (the second familial breast cancer ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12Q1/6869C12Q1/6886
CPCC12Q1/6806C12Q1/6869C12Q1/6886C12Q2600/156C12Q2600/166C12Q2535/101C12Q2535/122
Inventor 熊磊李福根谢正华王大磊
Owner 上海思路迪医学检验所有限公司
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