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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

Active Publication Date: 2017-02-15
3D BIOMEDICINE SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The conventional probe design method is that the probes are connected end to end, and there is no overlapping part. The disadvantage of this is that most fragments of the EGFR gene in the gene library can only be captured by a single probe, and the capture effect at the junction of the probes is poor. resulting in low capture efficiency
At the same time, the conventional probe design method has no special requirements on the probe length and Tm value, which leads to large differences among probes, and most probes cannot achieve the best capture effect at the same time under the same experimental conditions.

Method used

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  • Capture probe and kit used for high-flux sequencing detection of human circulating tumor DNA EGFR gene
  • Capture probe and kit used for high-flux sequencing detection of human circulating tumor DNA EGFR gene
  • Capture probe and kit used for high-flux sequencing detection of human circulating tumor DNA EGFR gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Preparation of Quality Control Products for Human Circulating Tumor DNA EGFR Gene Variation Detection

[0032] 1.1. Six commonly used 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. These six human tumor cell lines were cultured in a special medium, culture conditions: constant temperature at 37°C, 5% CO 2 , humidity 50%. Cultivate until the cell density reaches 80-90% of the area of ​​the culture dish, collect the cells in the logarithmic phase of growth, centrifuge at 1500g at 4°C for 5min, take the precipitate and discard the supernatant.

[0034] 1.3. Resuspend in pre-cooled PBS solution, centrifuge at 5000rpm at 4°C for 5min, ...

Embodiment 2

[0037] Example 2: Preparation of Human Circulating Tumor DNA EGFR Gene Variation 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 coding exon regions and exon-intron junction regions of the human EGFR gene; The capture probe is labeled with biotin; 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 together in the same ratio, and dilute the mixture to a working concentration of 1.5 PM (PM = picomole / liter), and store at -20°C.

[0040] 2.3. Separately pack the capture probe mixture and the quality control obtained in Example 1.

[0041] 2.4. Prepare instructions, outer packaging, assemble and seal.

[0042] 2.5. The amount of capture probe mixture used is 6 μL / 3 reactions, 12 μL / 6 reactions, 24 μL / 12 ...

Embodiment 3

[0043] Example 3: Sequencing detection of human circulating tumor DNA EGFR gene variation

[0044] The instrument used for sequencing in this example is the gene sequence automatic analyzer CN500.

[0045] The preparation method of the quality control product is the same as that in Example 1.

[0046] 3.1. Human cfDNA was extracted from 15 positive plasma samples, and quality control products were used directly without extraction.

[0047] 3.2. Library construction

[0048] 3.2.1. End Repair

[0049] Add 30ng of extracted cfDNA sample or quality control to 0.2ml PCR reaction tube, make up to 50μL with Low EDTA TE, vortex to mix, and centrifuge briefly. Add 1 μL of end-repair enzyme mixture and 6 μL of end-repair reaction buffer, vortex and mix briefly, and then incubate at 37°C for 5 minutes, then incubate at 65°C for 30 minutes, and then at 37°C for 5 minutes for end-repair. After the reaction, take out the PCR reaction tube and transfer all the products to a new 1.5mL ce...

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Abstract

The invention relates to a capture probe and kit used for high-flux sequencing detection of human circulating tumor DNA EGFR gene. The human circulating tumor DNA EGFR gene capture probe is a mixture of various probes, and the various probes respectively capture different target areas of the human EGFR gene. In a preferable enforcement scheme, the mixture of the various probes can capture all coding exon areas and exon intron boundary areas of the human circulating tumor DNA EGFR gene.

Description

[0001] field of invention [0002] The invention relates to the field of gene detection, in particular to a high-throughput sequencing-based probe and kit for detecting the capture of human circulating tumor DNAEGFR gene. Background technique [0003] There are free small fragments of DNA (cell-free DNA, cfDNA) in the blood, which come from dead cells. Usually dead cells will be removed, so the content of cfDNA is very low, usually 25ng cfDNA in 1ml plasma of a healthy person. The content of cfDNA in cancer patients is several times higher than normal, and part of it is ctDNA (circulating tumor DNA). The relative content of ctDNA is correlated with tumor burden and response to treatment, and can be used to identify driver genes, guide clinical treatment, monitor clinical treatment effects and cancer recurrence, reveal treatment resistance, and detect disease progression. In some respects the sensitivity of ctDNA method is even higher than traditional means. For example, tra...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q2521/501C12Q2525/191C12Q2535/122C12Q2525/204
Inventor 李福根熊磊金其煌覃灏
Owner 3D BIOMEDICINE SCI & TECH CO LTD
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