Breast cancer-related kinase mutation detection panel and application thereof

A breast cancer and kinase technology, applied in the field of kinase mutation detection panel, can solve the problems that cannot meet the needs of detection and diagnosis, and achieve the effect of improving positive detection rate, high throughput, and wide applicability

Pending Publication Date: 2020-02-18
FUDAN UNIV SHANGHAI CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current clinical application is only for the prediction of single or several kinase gene mutation points, which cannot meet the needs of actual clinical detection and diagnosis

Method used

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  • Breast cancer-related kinase mutation detection panel and application thereof
  • Breast cancer-related kinase mutation detection panel and application thereof
  • Breast cancer-related kinase mutation detection panel and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] RNA probe preparation:

[0036] The oligo pool of the target gene was obtained from Suzhou Synbio Biotechnology Co., Ltd., and diluted to 1ng / μl with 1×TE (PH8.0). The oligo sequence was amplified using Herculase II Fusion DNA Polymerase kit (Agilent). RNA transcription was performed with Ambion SP6 megascript kit (Thermo Fisher Scientific), and the probe preparation was completed. Store RNA probes at -80°C. Take part of the RNA probe library and dilute to 100ng / μl. The probes made by this method can be applied to 200-300 samples, which greatly reduces the cost of sequencing.

Embodiment 2

[0038] 1. DNA was extracted from a cancer tissue sample of a clinically diagnosed breast cancer patient (Fudan University Cancer Hospital), and the kits used were Tiangen Biochemical Technology (Beijing) Co., Ltd., TGuide Cells / Tissue Genomic DNAKit and Tiangen Biochemical Technology (Beijing) Co., Ltd. Take 200ng of DNA sample and dilute it to 50μl, transfer it into a 130μl Covaris interruption tube, use the Covaris instrument, and select the set 150-200bp condition to interrupt.

[0039]2. DNA sample library preparation: KAPA DNA Hyper Library Preparation Kit (Roche Sequencing) was used to prepare the library according to the kit instructions. Take 1ul of the library and use a microplate reader to detect the library concentration, the concentration is greater than 50ng / ul, and the total amount is not less than 750ng.

[0040] 3. Hybrid capture and sequencing

[0041] 1) Mix 95 μl of hybridization blocking reagent and a DNA library with a total volume of not less than 500ng...

Embodiment 3

[0050] Bioinformatics analysis: the original file is data in fastq format; use bwa for alignment, Haplotype Caller method to obtain germline mutation, MuTect2 for somatic mutation analysis, and ANNOVAR for annotation. The test results are shown in the table below.

[0051] Table 3 Detection results of leukocyte gene mutation

[0052]

[0053] *The detected germline variants and polymorphisms are listed in the table above after screening. The screening condition is that the ExAc or 1000 Genomes frequency is less than 0.05, and it is located in the exon region and causes amino acid changes, or it is located in the splice region.

[0054] Table 4 Detection results of tumor tissue gene mutations

[0055]

[0056]

[0057] Table 5 Detection results of polygenic copy number variation in tissues

[0058]

[0059] It can be seen that the mutations of the genes involved in the detection panel of the present invention can be detected and screened by the probes, which can ...

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PUM

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Abstract

The invention discloses a breast cancer-related kinase mutation detection panel. The detection panel comprises receptor tyrosine kinase (RTK), phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK) related genes, and an exon region and a part of intronic regions of a downstream gene. The invention further discloses a kit containing the detection panel. The detection panel can detect gene mutation guiding diagnosis and treatment more efficiently, is of great significance to clinical diagnosis and discovery of a target spot of target treatment, and can help lower gene detection cost of patients.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a detection panel for breast cancer-related kinase variation and an application thereof. Background technique [0002] Cancer is the leading cause of death and disability in the world today. Each tumor includes both genetic (germline) and tumor-specific (somatic) variation. In the past decade, high-throughput sequencing and bioinformatics technology have made great progress, and mutation information of a large number of germline and somatic genes has been collected. A large number of clinical practices have proved that the treatment of specific proteins that are mutated in cancer patients is effective. In order to achieve the goal of precision medicine, which is to give the right drug to the right patient, many laboratories have begun to apply next-generation sequencing technology in cancer genetic testing. [0003] The occurrence and development of breast c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/573
CPCG01N33/57415G01N33/573G01N2333/91215G01N2333/912
Inventor 邵志敏胡欣黄薇施锦绣
Owner FUDAN UNIV SHANGHAI CANCER CENT
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