Method for detecting BRCA1 and BRCA2 mutations based on next-generation sequencing technology

A next-generation sequencing technology and sequencing technology, applied in the field of molecular detection, can solve problems such as genome instability

Active Publication Date: 2020-10-20
ZHENYUE BIOTECHNOLOGY JIANGSU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Each failure of homologous recombination repair gene has th

Method used

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  • Method for detecting BRCA1 and BRCA2 mutations based on next-generation sequencing technology
  • Method for detecting BRCA1 and BRCA2 mutations based on next-generation sequencing technology
  • Method for detecting BRCA1 and BRCA2 mutations based on next-generation sequencing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1. Obtaining the gene sequence of tumor samples and blood cell samples of the kit of the application

[0063] 1. FFPE gDNA and BC gDNA fragmentation treatment

[0064] The concentration of FFPE DNA and BC DNA was diluted to 6ng / μL. Take 55μL for interruption. It is recommended to use CovarisM220 as the interrupter. Set the device parameters in the table below for fragmentation.

[0065]

[0066]

[0067] 2. Library construction

[0068] 2.1 End Repair & Add A:

[0069] 2.1.1 Configure the end repair & add A reaction system according to the following table, shake and mix, and centrifuge briefly.

[0070] Component Volume (μL) Fragmented DNAX End repair enzyme2 End repair buffer10 Nuclease-free water Top up 48 total capacity60

[0071] 2.1.2 Place the configured reaction system on the PCR machine, and perform PCR reaction according to the following table. Note: The temperature of the thermal lid of the PCR machine is set to 85°C.

[0072]

[0073] 2.2 Connector con...

Embodiment 2

[0174] Example 2. Detection of pathogenicity of homologous recombination genes in the kit of the application

[0175] 1. Identification of homologous recombination genes SNV and indel

[0176] Use Mutect2 software to analyze the gene-panel comparison results of the tumor samples and normal samples obtained in Example 1, identify somatic mutations and germline mutations in the tumor samples, and use annovar software to analyze the somatic cells identified by Mutect2 Mutations and germline mutations are annotated, and those that meet the following criteria are pathogenic mutations:

[0177] 1) The number of sequences covering the mutation site is greater than 200;

[0178] 2) The frequency of mutation is greater than 5%;

[0179] 3) Clinvar is recorded as a pathogenic mutation, or the mutation type is fs, truncate, splice.

[0180] 2. Detection of copy number variation of homologous recombination genes

[0181] Use cnvkit software to analyze the gene-panel comparison results of the tumor s...

Embodiment 3

[0182] Example 3. Calculation of the mutation feature score of the kit of the application

[0183] Somatic mutations are induced by different external or internal factors, including errors in the DNA replication mechanism, induction of internal or external factors, modification of DNA modification enzymes, or failure of DNA repair enzymes. Somatic mutations caused by different factors will have different combinations of mutation types, which are called mutation characteristics. It has been reported that Signature 3 in the mutation characteristics has a very strong correlation with BRCA1 and BRCA2 defects.

[0184] The SNV filtering result obtained in Example 2 was calculated using sigma software to obtain a mutation characteristic score related to homologous recombination.

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Abstract

The invention relates to a method and kit for detecting BRCA1 and BRCA2 mutations based on a next-generation sequencing technology, and belongs to the technical field of molecular detection. In orderto overcome the technical defect that the prior art fails to achieve full coverage of genes when detecting BRCA1 and BRCA2, the invention provides the method and kit for detecting BRCA1 and BRCA2 mutations based on the next-generation sequencing technology. In the method, after a to-be-detected gene is broken, an A joint is added, after corresponding PCR reaction, a designed probe is used for hybridization capture, and after a captured DNA library is amplified and sequenced and then evaluated by software to determine the BRCA1 and BRCA2 mutations. Compared with the prior art, the method achieves full gene coverage of BRCA1 and BRCA2, can obtain reliable data results and is suitable for promotion.

Description

Technical field [0001] The invention relates to a method and a kit for detecting BRCA1 and BRCA2 mutations based on second-generation sequencing technology, and belongs to the technical field of molecular detection. Background technique [0002] Homologous Recombination refers to the recombination that occurs between non-sister chromatids or between or within DNA molecules containing homologous sequences on the same chromosome. Homologous recombination allows the damaged chromosome to repair itself through the same DNA as another undamaged chromosome. This repair ensures the integrity of the genome. When a cell has a homologous recombination gene mutation, resulting in a homologous recombination deficiency (HRD), the cell cannot repair the DNA itself through homologous recombination. For example, the well-known breast cancer tumor-related genes BRCA1 and BRCA2 are homologous recombination proteins. When an individual's BRCA1 and BRCA2 genes are mutated, their lifetime risk of b...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2535/122C12Q2531/113C12Q2537/165C12Q2549/125C12Q2521/501C12Q2525/191
Inventor 张亚晰闫慧婷刘异倩陈敏俊吕红陈维之郑杉何骥杜波
Owner ZHENYUE BIOTECHNOLOGY JIANGSU CO LTD
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