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Quality control method for detecting human BRCA1/2 genovariation based on high-throughput sequencing and reagent kit

A gene variation and kit technology, applied in the field of genetic detection, can solve the problems of low detection efficiency, no obvious site specificity, and failure to meet the detection requirements well, so as to improve efficiency and reduce detection quality control costs Effect

Active Publication Date: 2017-02-08
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 for detecting human BRCA1/2 genovariation based on high-throughput sequencing and reagent kit
  • Quality control method for detecting human BRCA1/2 genovariation based on high-throughput sequencing and reagent kit
  • Quality control method for detecting human BRCA1/2 genovariation based on high-throughput sequencing and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Preparation of Quality Control Products for Human BRCA1 / 2 Gene Variation Detection

[0036] 1.1. Four commonly used human tumor cell lines HCC1599, HCT-15, Jurkat, and NCI-H720 that can be stably passaged were purchased from ATCC.

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

[0038] 1.3. Dilute the purified genomic DNA of each cell line with Tris-EDTA buffer solution to 100±5ng / μL, the appearance is transparent liquid, no impurities visible to the naked eye, and the purity is 1.9>OD260 / 280>1.7, Get quality control material DNA.

[0039] 1.4. The genomic ...

Embodiment 2

[0041] Embodiment 2: Preparation of human BRCA1 / 2 gene variation 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 coding exon regions of the human BRCA1 / 2 gene and exon-intron junctions 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 Variation Detection Hybridization Capture Probe Sequence

[0044]

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[0050]

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[0065]

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[0070]

[0071]

[0072]

[0073] 2.2. Mix all the above-mentioned capture probes with the same mass, and dilute the mixture to a working concentration of 1...

Embodiment 3

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

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

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

[0080] 3.1. Human genomic DNA was extracted from 20 positive blood samples, and the positive quality control was used directly without extraction.

[0081] 3.2. Fragmentation of DNA samples and quality controls

[0082] Take 200ng of each human genomic DNA sample, and 200ng of the quality control substance. If it is less than 50μL, it can be made up to 50μL with PCR-gradewater, vortexed and mixed, and centrifuged briefly. Then all the solution was transferred to a microtube, and the fragmentation program with a fragment length of 200-300bp was selected to fragment the genomic DNA sample and quality control DNA. Take 5 μL of the fragmented sample for agarose gel electrophoresis.

[0083] 3.3. Library Construction

[0084] 3.3.1....

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Abstract

The invention relates to a quality control method for detecting human BRCA1 / 2 genovariation based on high-throughput sequencing, a reagent kit, and an application thereof. In the quality control method, a plurality of genome DNAs of a human tumor cell line with positive BRCA1 / 2 genovariation are extracted, and are determined by means of a Sanger sequencing method, wherein a variation positive locus is used as a positive comparison locus while a wild type locus is a negative comparison locus; the genome DNAs are mixed at a certain ratio to obtain a quality control product that can be used for detecting human BRCA1 / 2 genovariation through high-throughput sequencing. The reagent kit disclosed by the invention comprises the quality control product for detecting human BRCA1 / 2 genovariation.

Description

[0001] field of invention [0002] The invention relates to the field of gene detection, in particular to a quality control method and a kit for detecting human BRCA1 / 2 gene variation based on high-throughput sequencing. Background technique [0003] Breast cancer is one of the most common malignant tumors that seriously affects women's health and even threatens their lives. According to statistics, its incidence rate accounts for 7-10% of various malignant tumors in the whole body. The incidence of breast cancer is often related to heredity. In addition, between the ages of 40 and 60, the incidence rate of women before and after menopause is higher. [0004] Breast cancer can be divided into sporadic and hereditary two categories. 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 ca...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
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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