Method for capturing genetic colorectal cancer genome target sequence

A technology of colorectal cancer and genomics, applied in biochemical equipment and methods, combinatorial chemistry, microbial measurement/testing, etc., can solve the problem of not being able to screen multiple key areas of gene mutation load in a targeted manner and not well satisfying individualization problems such as specific user needs, long single-sample analysis time, etc., to achieve the effect of reducing detection cost and sequencing data volume, reducing non-specific capture, and improving sequencing depth

Inactive Publication Date: 2021-08-10
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] The purpose of the present invention is to provide a method for capturing the genomic target sequence of hereditary colorectal cancer, so as to solve the problem that the existing high-throughput sequencing method for capturing the genomic target sequence proposed in the background technology cannot screen the gene mutation load in a targeted manner. Targeted design of high-efficiency liquid-phase target gene capture probes and supporting library construction kits in key areas, resulting in a large amount of sequencing data and long single-sample analysis time, which cannot well meet individual user needs
[0011] In order to realize the above-mentioned method for capturing the genomic target sequence of hereditary colorectal cancer, the existing high-throughput sequencing method for capturing the genomic target sequence proposed in the background technology cannot screen multiple key regions of gene mutation load in a targeted manner. The design of high-efficiency liquid-phase target gene capture probes and supporting library construction kits results in a large amount of sequencing data and a long time for single-sample analysis, which cannot well meet individual user needs. The present invention provides the following technical solutions:

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  • Method for capturing genetic colorectal cancer genome target sequence
  • Method for capturing genetic colorectal cancer genome target sequence
  • Method for capturing genetic colorectal cancer genome target sequence

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Preparation of detection quality control products for high-throughput capture sequencing of hereditary colorectal cancer gene mutations.

[0050] (1) Two commonly used stable human colorectal cancer cell lines HCT-116 and HCT-15 were purchased from ATCC;

[0051] (2) Adopt fetal calf serum medium (provided by Thermo Fisher Company) to cultivate these two strains of human tumor cell lines, culture conditions: 37 ℃ constant temperature, 5% CO , humidity 50%; Cultivate until the cell density reaches the area of ​​the culture dish 80- Passage at 90%, collect in the logarithmic phase of cell growth, centrifuge at a speed of 800-1000r / min to take the precipitate, extract the genomic DNA of each cell line separately, and purify and elute through the column;

[0052] (3) Dilute the purified genomic DNA of each cell line to 100±5ng / μL with Tris-EDTA buffer solution. The appearance is a transparent liquid without visible impurities, and the purity is 1.9>OD260 / 280>1.7 , to obtai...

Embodiment 2

[0056] capture probe design

[0057] Select the key region of hereditary colorectal cancer genome mutation, design and synthesize multiple capture probes according to the key region, and some examples of the key region are shown in Table 2; examples of the sequence design of the capture probe are as SEQ ID NO:1~ 2641; the length of the capture probe is 100bp, and the capture probes are connected end to end, covering the exon region and exon-intron junction region of each target region; all probes are labeled with biotin, and the The capture probes are 2641 probe mixtures, and the design method of the capture probes is:

[0058] (1) Determine the exon region and exon-intron junction region of each target region according to the human whole genome standard sequence HG19 provided by the public data website NCBI;

[0059] (2) Starting from the exon region in each region and the first exon in the exon-intron junction region, design the capture probe according to the standard seque...

Embodiment 3

[0066] Preparation of Genomic Mutation Capture Sequencing Kit for Hereditary Colorectal Cancer

[0067] (1) Design and synthesize the capture probes of the target regions shown in Example 2;

[0068] (2) Mix the designed capture probes with the same mass, dilute the mixture to a working concentration of 25ng / μL, and store at -20°C;

[0069] (3) Separately pack the capture probe mixture and the quality control obtained in Example 1.

[0070] (4) Prepare instructions, outer packaging, assemble and seal.

[0071] (5) The amount of capture probe mixture used is 6 μL / 3 reactions, 12 μL / 6 reactions, 24 μL / 12 reactions, 48 ​​μL / 24 reactions; the amount of quality control products is 30ng-50ng / reaction.

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Abstract

The invention belongs to the technical field of gene detection, and particularly discloses a method for capturing a genetic colorectal cancer genome target sequence, wherein the sequence of a capture probe adopted by the method is as shown in SEQ ID NO: 1-2641, and the capture probe covers a region of 80-300Kb, preferably 80-100Kb, of the genome, and the Tm value of the capture probe is 62-76 DEG C. The probe designed and synthesized by the invention can efficiently and completely capture a coding exon region and an exon-intron junction region of a human genome target region, reduces non-specific capture, is uniform in hybridization temperature, greatly reduces the detection cost and the sequencing data volume, shortens the single sample analysis time, and meets the personalized user requirements; the method is especially suitable for screening of high-risk population of hereditary colorectal cancer and public physical examination markets.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method for capturing genome target sequences of hereditary colorectal cancer. Background technique [0002] Colorectal cancer is the third most common malignant tumor in the world, and its mortality rate is the second highest, with approximately 1.2 million new patients each year. In China, the morbidity and mortality of colorectal cancer are increasing year by year. About 1 / 3 of patients have a family genetic background, and 5% to 6% of patients can be diagnosed as hereditary colorectal cancer. The awareness of colorectal cancer is insufficient. At present, in addition to several specialized tumor hospitals and a few general hospitals that have carried out related work, there is still a lack of systematic registration and screening of hereditary colorectal cancer. Therefore, the actual diagnosis rate of hereditary colorectal cancer in China is still low. Hereditary co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6806C12Q1/6869C40B50/06
CPCC12Q1/6886C12Q1/6806C12Q1/6869C40B50/06
Inventor 王征王琳王国斌徐鲁明路小欢雷世俊刘扬王星月
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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