Capturing probe and kit for high-throughput sequencing detection of human circulating tumor DNA KRAS gene

A capture probe, high-throughput technology, applied in DNA/RNA fragment, recombinant DNA technology, microbial determination/inspection, etc., can solve the problem that the probe cannot achieve the capture effect at the same time, the capture effect at the probe junction is poor, and the capture problems such as low efficiency, to achieve the effect of good sequencing data quality, improved capture capability, and high capture efficiency

Active Publication Date: 2017-03-22
3D BIOMEDICINE SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 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 KRAS 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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  • Capturing probe and kit for high-throughput sequencing detection of human circulating tumor DNA KRAS gene
  • Capturing probe and kit for high-throughput sequencing detection of human circulating tumor DNA KRAS gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation of Quality Control Products for Human Circulating Tumor DNA KRAS Gene Variation Detection

[0030]1.1. Seven commonly used stable human tumor cell lines HOP-62, HCT-15, AGS, NCI-H1573, COR-L23, NCI-H1355 and SW1417 were purchased from ATCC. The genomic DNA of each cell line was sequenced by the Sanger method, and the heterozygous and homozygous mutation sites confirmed by the Sanger method were used as positive control sites. It has been verified that there are 6 positive mutation sites in total.

[0031] 1.2. The seven human tumor cell lines were cultured in a special medium, culture conditions: 37°C constant temperature, 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.

[0032] 1.3. Resuspend in pre-cooled PBS solution, centrifuge at 5000rpm at 4°...

Embodiment 2

[0035] Example 2: Preparation of Human Circulating Tumor DNA KRAS Gene Variation Detection Kit

[0036] 2.1. Design and synthesize multiple capture probes for different target regions on the human circulating tumor DNA KRAS gene. The collection of all capture probes can cover all coding exon regions and exon-intron junction regions of the human KRAS gene; The capture probe is labeled with biotin; the sequence of the capture probe is shown in SEQ ID NO: 1-37 in the sequence listing.

[0037] 2.2. Mix all the capture probes shown in SEQ ID NO: 1-37 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.

[0038] 2.3. Separately pack the capture probe mixture and the quality control obtained in Example 1.

[0039] 2.4. Prepare instructions, outer packaging, assemble and seal.

[0040] 2.5. The amount of capture probe mixture used is 6 μL / 3 reactions, 12 μL / 6 reactions, 24 μL / 12 re...

Embodiment 3

[0041] Example 3: Sequencing detection of human circulating tumor DNA KRAS gene variation

[0042] The instrument used for sequencing in this example is NextSeq CN500, an automatic gene sequence analyzer.

[0043] The preparation method of the quality control product is the same as that in Example 1.

[0044] 3.1. Human cfDNA was extracted from 10 positive plasma samples, and quality control products were used directly without extraction.

[0045] 3.2. Library construction

[0046] 3.2.1. End Repair

[0047] 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 ...

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Abstract

The invention relates to a capturing probe and kit for high-throughput sequencing detection of a human circulating tumor DNA KRAS gene. The human circulating tumor DNA KRAS gene capturing probe is a mixture of multiple kinds of probes which can capture different target regions on the human KRAS gene. In an optimized embodiment, the set of the probes can capture all coded exon regions and exon and intron boundary regions of the human circulating tumor DNA KRAS 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 DNA KRAS 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, tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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