Capture sequencing probe and application thereof
A capture probe and sequencing technology, applied in the field of capture sequencing probes, can solve the problems of not being able to screen multiple key regions of gene mutation load, not being able to meet individual user needs, and taking a long time for single-sample analysis , to achieve the effects of reducing detection cost and sequencing data volume, shortening single-sample analysis time, and reducing non-specific capture
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Embodiment 1
[0106] Preparation of quality control products for high-throughput capture sequencing of colorectal cancer individualized drug-related gene mutations:
[0107] (1) Two commonly used stable human colorectal cancer cell lines HCT-116 and HCT-15 were purchased from ATCC;
[0108] (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;
[0109] (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 , t...
Embodiment 2
[0113] Design of capture probes: select the key regions of genomic mutations related to colorectal cancer individualized medicine, design and synthesize multiple capture probes according to the key regions, some examples of the key regions are shown in Table 2; the designed sequences of the capture probes are shown in Table 2. For example, as shown in SEQ ID NO: 1-2909; 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 All have biotin labels, and the capture probe is a mixture of 2909 probes, and the design method of the capture probe is:
[0114] (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;
[0115] (2) Starting from the exon region in each region and the first exon in the exon-intron junction region, design ...
Embodiment 3
[0121] Preparation of colorectal cancer individualized drug-related genome mutation capture sequencing probe kit
[0122] (1) Design and synthesize the capture probes of the target regions shown in Example 2;
[0123] (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;
[0124] (3) Separately pack the capture probe mixture and the quality control obtained in Example 1;
[0125] (4) Prepare instructions, outer packaging, assemble and seal;
[0126] (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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