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A probe library for detecting sensitive and drug-resistant mutations of fgfr family gene inhibitors and its application

A technology of gene suppression and FGFR3, applied in the determination/inspection of microorganisms, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the difference in accuracy, affect the precise use, and detect gene point mutations and small fragment insertions and deletions at the same time , copy number amplification and fusion, etc., to achieve the effects of high detection reaction throughput, broad application prospects, and compressed time and cost

Active Publication Date: 2021-10-12
QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this product can only detect the FGFR1 gene, and cannot detect the mutations of other genes in the FGFR family
[0005] At present, gene mutations can be detected by PCR method or high-throughput sequencing method, but the PCR method cannot simultaneously screen all coding regions of the FGFR gene, and cannot detect gene point mutations, Small fragment insertion and deletion, copy number amplification and fusion and other variant forms have limitations in detection throughput and cannot meet the needs of practical applications
The hybrid capture high-throughput sequencing method enriches the library fragments related to the target gene, combined with large-scale sequencing analysis, it is not limited by throughput, and due to the error tolerance of probe hybridization capture, it can simultaneously enrich variant and wild-type fragments, It can detect multiple variant forms at the same time to realize variant screening, but the detection of copy number amplification and fusion puts forward high requirements for the design of probes and the optimization of hybridization capture system, and the accuracy of detection of different variant forms There are also large differences, which directly affect the precise clinical use of FGFR inhibitor drugs

Method used

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  • A probe library for detecting sensitive and drug-resistant mutations of fgfr family gene inhibitors and its application
  • A probe library for detecting sensitive and drug-resistant mutations of fgfr family gene inhibitors and its application
  • A probe library for detecting sensitive and drug-resistant mutations of fgfr family gene inhibitors and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0311] This embodiment provides a probe library for detecting sensitive and drug-resistant mutations of FGFR family gene inhibitors. The probe library for detecting sensitive and drug-resistant mutations of FGFR family gene inhibitors includes:

[0312] Probe combinations for detecting exons and UTR regions of FGFR1, FGFR2, FGFR3 and FGFR4;

[0313] Probe combinations for detecting introns 1, 3 and 8 of FGFR1, introns 2 and 17 of FGFR2, and introns 7 and 17 of FGFR3;

[0314] And a combination of probes for detecting the sequences on both sides of the breakpoint of the FGFR family gene fusion gene;

[0315] The type of variation includes any one or a combination of at least two of point mutation, small fragment insertion, deletion, fusion or copy number amplification.

[0316] The probe library includes detection fusion product sequences for the sequences on both sides of the above-mentioned fusion gene breakpoints, and probe pairs that are completely complementary to the seq...

Embodiment 2

[0332] This embodiment provides a kit for detecting sensitive and drug-resistant mutations of FGFR family gene inhibitors, the kit for detecting sensitive and drug-resistant mutations of FGFR family gene inhibitors includes the detection of FGFR family gene inhibitors described in Example 1 Probe libraries for sensitive and drug-resistant variants;

[0333] The kit for detecting sensitive and drug-resistant mutations of FGFR family gene inhibitors also includes genomic pre-library construction reagents, adapters, hybridization capture reagents, hybridization product amplification reagents, and streptavidin-affinity magnetic beads. The specific components are shown in Table 2 shown.

[0334] Table 2

[0335]

[0336] The kit for detecting FGFR family gene inhibitor-sensitive and drug-resistant mutations of the invention is easy to operate and has high detection efficiency, and can simultaneously detect mutations of FGFR family genes in multiple samples, with accurate result...

Embodiment 3

[0338] In this example, using the kit for detecting sensitive and drug-resistant mutations of FGFR family gene inhibitors prepared in Example 2, three standard products (HD729, CBP10422 and CBP40037), 2 Two kinds of cell lines SW780 and CBP20188R and 4 clinical samples with known mutations were tested. The detection flow chart is as follows figure 1 As shown, it specifically includes the following steps:

[0339] (1) Extract the DNA in the sample;

[0340] According to the instructions of the QIAamp DNA Mini Kit extraction kit, DNA was extracted and quantified by Qubit, requiring tissue DNA ≥ 100 ng.

[0341] (2) Prepare the genome pre-library:

[0342] ① Enzyme digestion and end repair of the obtained sample DNA

[0343] The reaction system is as follows:

[0344] Component Volume (μL)

[0345] Buffer 5

[0346] Digestion and Repair Enzymes 10

[0347] DNA template 35

[0348] Total volume 50

[0349] Shake the above mixture thoroughly and centrifuge, and then run ac...

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Abstract

The invention provides a probe library for detecting FGFR family gene inhibitor sensitivity and drug resistance variation and its application, the probe library for detecting FGFR family gene inhibitor sensitivity and drug resistance variation includes: detecting FGFR1, FGFR2, FGFR3 Combines probes with exons and UTR regions of FGFR4; detects introns 1, 3, and 8 of FGFR1, introns 2 and 17 of FGFR2, and introns 7 and 17 of FGFR3. A probe combination; and a probe combination for detecting sequences on both sides of the FGFR family gene fusion gene breakpoint; the type of variation includes any one or at least two of point mutation, small fragment insertion, deletion, fusion or copy number amplification. combination of species. The invention also provides a kit for detecting the sensitivity and drug resistance variation of the FGFR family gene inhibitor and a using method thereof, which have extremely high application value.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a probe library for detecting sensitive and drug-resistant mutations of FGFR family gene inhibitors and an application thereof. Background technique [0002] Receptor tyrosine kinases (RTKs) are a very important family of protein kinases in the human body. They are the largest type of enzyme-linked receptors, which can bind to ligands and phosphorylate tyrosine residues of target proteins. At present, 58 receptor tyrosine kinases are known, which can be divided into 20 subfamilies according to the structure of extracellular and extracellular regions. The cell signaling pathway mediated by RTK plays an important role in the development of the body and the maintenance of the homeostasis of the cell. Abnormal activation of RTKs can lead to various pathological changes, especially in cancers, thus promoting the development of related RTK signaling inhibitory drugs...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q1/6886C12Q2600/106C12Q2600/156C12Q2525/191C12Q2531/113C12Q2565/519
Inventor 顾凯丽冯瑞杰邓啸韩竹青张亚飞
Owner QIAGEN SUZHOU TRANSLATIONAL MEDICINE CO LTD