A method and kit for detecting brca1 and brca2 mutations based on next-generation sequencing technology

A second-generation sequencing technology and sequencing technology, applied in the field of molecular detection, can solve problems such as genome instability, and achieve the effects of increasing sensitivity and specificity, good uniformity, and high capture efficiency

Active Publication Date: 2021-10-19
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 the potential to lead to the occurrence of genome instability

Method used

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  • A method and kit for detecting brca1 and brca2 mutations based on next-generation sequencing technology
  • A method and kit for detecting brca1 and brca2 mutations based on next-generation sequencing technology
  • A method and kit 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 Sequences of the Tumor Samples and Blood Cell Samples of the Kit of the Application

[0063] 1. Fragmentation of FFPE gDNA and BC gDNA

[0064] The concentration of FFPE DNA and BC DNA was diluted to 6ng / μL. Take 55 μL for fragmentation. CovarisM220 is recommended for the fragmentation instrument. Set the equipment parameters as shown 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 table, vortex to mix, and briefly centrifuge.

[0070] components Volume (μL) Fragmented DNA X end repair enzyme 2 end repair buffer 10 nuclease free water make up 48 total capacity 60

[0071] 2.1.2 Put the configured reaction system on the PCR instrument, and perform the PCR reaction according to the table below. Note: The thermal lid temperature of the...

Embodiment 2

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

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

[0176] Use Mutect2 software to analyze the comparison results of the tumor sample obtained in Example 1 and the normal sample next-generation sequencing gene-panel, identify the somatic mutation and germline mutation in the tumor sample, and use the annovar software to analyze the somatic cell identified by Mutect2 Mutations and germline mutations are annotated, and those that meet the following criteria are disease-causing 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) It is recorded as a pathogenic mutation in Clinvar, or the mutation type is fs, truncate, or splice.

[0180] 2. Homologous recombination gene copy number variation detection

[0181] Use the cnvkit sof...

Embodiment 3

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

[0183] Somatic mutations are induced by different external or internal factors, including errors in 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 is the so-called mutation signature. It has been reported that Signature 3 in the mutation signature has a very strong correlation with BRCA1 and BRCA2 defects.

[0184] For the filtering results of the SNVs obtained in Example 2, the sigma software was used to calculate the score of the mutation feature related to homologous recombination.

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Abstract

The invention relates to a method and a kit for detecting mutations of BRCA1 and BRCA2 based on next-generation sequencing technology, and belongs to the technical field of molecular detection. In order to overcome the technical insufficiency that the prior art fails to achieve full coverage of genes when detecting BRCA1 and BRCA2, the present invention provides a method and kit for detecting BRCA1 and BRCA2 mutations based on next-generation sequencing technology. In this method, the gene to be tested is After breaking, add A linker, carry out the corresponding PCR reaction, use the designed probe for hybridization capture, amplify the captured DNA library, perform library sequencing, and use software to evaluate to determine BRCA1 and BRCA2 mutations. Compared with the existing technology, this method achieves full gene coverage of BRCA1 and BRCA2, and the data results are reliable, which is suitable for promotion.

Description

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

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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