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Standard substance for extensive oncogene detection and preparation method and application thereof

A tumor gene and detection standard technology, applied in the field of pan-tumor gene detection standards and their preparation, which can solve the problems of limited number of driver genes and lack of consensus.

Active Publication Date: 2020-06-26
菁良科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to different detection and analysis methods, there are risks of false positive and false negative detection, such as next generation sequencing (Next Generation Sequencing, NGS), the detection sensitivity is related to the depth of sequencing. Generally speaking, NGS is used in the detection of tumor somatic mutations. , the detection sensitivity is 10%, the number of known tumor-related driver genes is limited, and the relationship between disease phenotype and genotype still depends on the interpretation of biological information, and the standardization and quality control of NGS for tumor cell mutation detection have not yet been formed consensus

Method used

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  • Standard substance for extensive oncogene detection and preparation method and application thereof
  • Standard substance for extensive oncogene detection and preparation method and application thereof
  • Standard substance for extensive oncogene detection and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Gene editing and single cell cloning

[0079] The gene editing method described in the present invention can obtain most of the clinically popular tumor gene mutation sites, such as epidermal growth factor receptor gene (EGFR), etc., as shown in the following table:

[0080]

[0081] In one or more embodiments of the present invention, the specific method is:

[0082] a. For the target site of the target gene, design a specific guide RNA (guide RNA, gRNA), and construct the expression vector of the guide RNA. At the same time, single-stranded DNA is designed and synthesized to be used as a template for gene editing and repair.

[0083] b. Co-transfect the expression vector of the guide RNA and the single-stranded DNA into the target cells.

[0084] c. When the cells are in good condition, perform single cell cloning.

[0085] d. After the formation of cell clones, some clones were taken for sanger sequencing to confirm that the gene editing was successfu...

Embodiment 2

[0092] Example 2 Preparation of Pan-tumor Gene Detection Standard Samples to be Detected

[0093] (1) Genomic DNA extraction

[0094] Using Promega 16Cell LEV Purification Kit, Cat. No. AS1140 and the usage method in its instruction manual are used to extract gDNA.

[0095] (2) Concentration determination

[0096] ① Use a spectrophotometer to measure the DNA concentration of the extracted product.

[0097] ② Concentration determination should be repeated three times in a row, and the following conditions should be met:

[0098] Concentration: average concentration 20.0ng / μL≤x≤60.0ng / μL;

[0099] OD260 / 280: The measured value is 1.8≤x≤2.0, which is qualified;

[0100] OD260 / 230: The measured value is 1.5≤x≤5.0, and it is determined to be qualified.

[0101] (3) mixed

[0102] ①Mix the gDNA extracted from the cell pellet with the same code and batch number, and confirm before mixing:

[0103] The gDNA to be mixed is extracted from cell pellets with the same code and bat...

Embodiment 3

[0112] Example 3 Detection of Pan-tumor Gene Detection Standard Samples to be Detected

[0113] (1) ddPCR detection

[0114] The pan-tumor gene detection standard sample obtained in Example 2 is tested for the gene frequency of the gene loci in the table below by ddPCR, and the original mutation frequency of the raw material is confirmed by ddPCR (BIO-RAD QX200 platform) to obtain ddPCR verification Mutation site variation information.

[0115]

[0116] The specific ddPCR experimental procedure is as follows:

[0117]① Configure the reaction master mix according to the reaction components in the table below, and make 2 to 4 replicate wells for each sample at each site. Considering the loss of pipetting, configure 1.1 reaction mixtures for each amplification well at each site. quantity. See the table below for detailed configuration components and their dosage:

[0118]

[0119] ② Droplets occur. Add the above-mentioned premix to the middle hole of the droplet genera...

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Abstract

The invention provides a standard substance for extensive oncogene detection. The standard substance includes 13 mutation sites verified by Droplet Digital PCR (ddPCR) and 500X high-throughput whole exome sequencing verified site information, has more than 700 variation sites of over 330 genes, also includes common structural variations of cancer genomes, such as common gene SNV, base insertion deletion and the like, has corresponding mutation site frequencies within a range of 1% to 100%, and is wide in application scene and platform. The standard substance can be used for evaluating stability, specificity and sensitivity of the working flow from sample extraction to biological information analysis, evaluating each sample treatment method and detecting performance differences among platforms. The standard substance belongs to a major innovation in the industry, and has wide application prospect and great industrial application value.

Description

technical field [0001] The invention relates to the field of tumor gene detection, in particular to a standard product for pan-tumor gene detection and its preparation method and application. Background technique [0002] In recent years, gene detection for individualized treatment of tumors has been widely used clinically. By detecting nucleotide polymorphisms, insertions or deletions, and copy number variations of biomarker genes in biological samples of tumor patients, they can predict drug efficacy and evaluate prognosis. , to guide clinical individualized treatment. However, due to different detection and analysis methods, there are risks of false positive and false negative detection, such as next generation sequencing (Next Generation Sequencing, NGS), the detection sensitivity is related to the depth of sequencing. Generally speaking, NGS is used in the detection of tumor somatic mutations. , the detection sensitivity is 10%, the number of known tumor-related driver...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6806C12Q1/6886
CPCC12Q1/6806C12Q1/6886C12Q2600/166C12Q2531/113C12Q2563/159C12Q2535/122C12Q2537/165
Inventor 韦良慎李菁华梁达超林东旭魏孝林
Owner 菁良科技(深圳)有限公司
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