Capture probe and kit used for high-flux sequencing detection of human circulating tumor DNA EGFR gene
A capture probe, high-throughput technology, used in DNA/RNA fragments, recombinant DNA technology, microorganism determination/inspection, etc. problems such as low efficiency, to achieve the effect of good sequencing data quality, improved capture capability, and high capture efficiency
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Example Embodiment
[0031] Example 1: Preparation of quality control materials for detection of human circulating tumor DNA EGFR gene mutation
[0032] 1.1. Six commonly used and stable human tumor cell lines A549, NCI-H720, NCI-H1650, NCI-H1975, SW48 and SW1417 were purchased from ATCC. The genomic DNA of each cell line was sequenced by digital PCR, and the heterozygous and homozygous variant sites confirmed by the Sanger method were used as positive control sites. It has been verified that there are 12 positive mutation sites in total.
[0033] 1.2. The six human tumor cell lines were cultivated with a special medium, and the culture conditions: constant temperature of 37°C, 5% CO 2 , Humidity 50%. Cultivate until the cell density reaches 80-90% of the culture dish area and pass it down. Collect in the logarithmic phase of cell growth. Centrifuge at 1500g at 4°C for 5min to collect the pellet and discard the supernatant.
[0034] 1.3. Resuspend in pre-cooled PBS solution, centrifuge at 5000 rpm at 4...
Example Embodiment
[0037] Example 2: Preparation of human circulating tumor DNA EGFR gene mutation detection kit
[0038] 2.1. Design and synthesize multiple capture probes for different target regions on the human circulating tumor DNA EGFR gene. The collection of all capture probes can cover all the coding exon regions and exon-intron junction regions of the human EGFR gene; The capture probe has a biotin label; the sequence of the capture probe is shown in SEQ ID NO: 1-195 in the sequence listing.
[0039] 2.2. Mix all the capture probes shown in SEQ ID NO: 1-195 in the sequence listing in the same proportion, and dilute the mixture to a working concentration of 1.5 PM (PM = picomoles / liter) and store at -20°C.
[0040] 2.3. Separate the capture probe mixture and the quality control product obtained in Example 1.
[0041] 2.4. Prepare instructions, outer packaging, and assemble and seal.
[0042] 2.5. The amount of capture probe mixture is 6μL / 3 reaction, 12μL / 6 reaction, 24μL / 12 reaction, 48μL / 24 rea...
Example Embodiment
[0043] Example 3: Sequencing detection of human circulating tumor DNA EGFR gene mutation
[0044] The instrument used for sequencing in this embodiment is an automatic gene sequence analyzer CN500.
[0045] The preparation method of the quality control product is the same as in Example 1.
[0046] 3.1. Extract human cfDNA from 15 positive plasma samples, and use quality control products directly without extraction.
[0047] 3.2. Library construction
[0048] 3.2.1. End repair
[0049] 0.2ml PCR reaction tube, add 30ng of extracted cfDNA sample or quality control material, make up to 50μL with Low EDTA TE, shake and mix, and centrifuge briefly. Add 1 μL end repair enzyme mixture and 6 μL end repair reaction buffer respectively, shake and mix briefly, and then centrifuge at 37°C for 5 min, then at 65°C for 30 min and 37°C for 5 min to perform end repair. After the reaction, take out the PCR reaction tube and transfer all the products to a new 1.5mL centrifuge tube; add 108μL of nucleic a...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap