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Capturing probe for detecting human genome mutation load based on high-throughput sequencing and application thereof

A technology for capturing probes and mutation loads, applied in the field of genes, can solve the problems of large data volume, high detection cost, and long analysis time, and achieve the effects of reducing detection costs, uniform hybridization temperature, and shortening analysis time

Pending Publication Date: 2017-12-19
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 technical problem solved by the present invention is: the existing mutation load detection method is whole exome sequencing, the detection cost of the method is high, the amount of data is large, and the analysis time is long

Method used

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  • Capturing probe for detecting human genome mutation load based on high-throughput sequencing and application thereof
  • Capturing probe for detecting human genome mutation load based on high-throughput sequencing and application thereof
  • Capturing probe for detecting human genome mutation load based on high-throughput sequencing and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Preparation of Quality Control Products for Human Genome Mutation Burden Detection

[0056] (1) Two commonly used human tumor cell lines HCC1937 and HCT-15 that can be stably passaged were purchased from ATCC;

[0057] (2) The two strains of human tumor cell lines were cultured using fetal bovine serum medium (provided by Thermo Fisher Company), culture conditions: 37°C constant temperature, 5% CO 2 , humidity 50%; culture until the cell density reaches 80-90% of the area of ​​the culture dish, then subculture, collect in the logarithmic phase of cell growth, centrifuge at a speed of 800-1000r / min to take the precipitate, and extract the genomic DNA of each cell line separately, And column purification, elution;

[0058] (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 obtain quality control material D...

Embodiment 2 8

[0063] Example 2 0.8Mb mutation load assessment region capture probe design

[0064] Select the key region of human genome mutation load, design and synthesize multiple capture probes according to the key region, the key region is as shown in Table 2; the capture probe design sequence example is shown in SEQ ID NO:1-275; the The length of the capture probe is 120bp, and the capture probe is connected end to end, covering the exon region and exon-intron junction region of the 0.8Mb key region; all probes are labeled with biotin, and the capture probe For 6686 probe mixtures, the design method of the capture probes is:

[0065] (1) Determine the exon region and exon-intron junction region of each target region (see Table 2) according to the human genome standard sequence HG19 (Human Genome19) provided by the public data website NCBI;

[0066] (2) Starting from the exon region in each region and the first exon in the exon-intron junction region, design the capture probe accordin...

Embodiment 3 2

[0071] Example 3 1.2Mb mutation load assessment region capture probe design

[0072] Select the key region of human genome mutation load, design and synthesize multiple capture probes according to the key region; the key region is as shown in Table 3; the capture probe design sequence example is shown in SEQ ID NO:276-550; the The length of the capture probe is 120bp; the capture probe is connected end to end, covering the 1.2Mb key region of the exon region and the exon-intron junction region; all probes are biotin-labeled; the capture probe is 10333 probe mixtures; the capture probe design method is the same as in Example 2.

[0073] Table 3 key areas

[0074]

[0075]

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Abstract

The invention relates to the field of gene detection, in particular to a capturing probe for detecting human genome mutation load based on high-throughput sequencing and application thereof. The capturing probe for detecting the human genome mutation load based on high-throughput sequencing, provided by the invention, comprises a capturing probe 1, a capturing probe 2 and a capturing probe 3; the sequences of the capturing probe 1 are shown as SEQ ID NO. 1-275; the sequences of the capturing probe 2 are shown as SEQ ID NO. 276-550; the sequences of the capturing probe 3 are shown as SEQ ID NO. 551-825; the probe designed and synthesized by the invention can capture an encoding exon area in a human genome target area and an exon and intron junction area, the nonspecific capture is reduced, and meanwhile, the hybrid temperature is uniform. As the capturing probe provided by the invention is adopted for capturing the human genome target area, the sequencing depth is significantly improved, so that mutation load test results are highly correlated with WES, the detection cost and the sequencing data volume are greatly reduced, and the single sample analysis time is shortened.

Description

technical field [0001] The invention relates to the field of genes, in particular to a high-throughput sequencing-based detection of human genome mutation load capture probes and applications. Background technique [0002] In recent years, with the continuous development of molecular biology, immunology, biochemistry, clinical medicine, laboratory medicine and other related disciplines, immunotherapeutic tumor drugs have shown broad application prospects. More and more immuno-oncology drugs have been developed and applied clinically, benefiting more patients. However, there will be significant individual differences in the clinical application of immuno-oncology drugs, and different patients have different drug sensitivities to the same drug. For example, only about 5% of patients with colorectal cancer are sensitive to immunosuppressant drugs, while the rest of the patients have no ideal drug response after treatment. This phenomenon has also been observed in other cancer...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6888C12Q2600/156C12Q2535/122
Inventor 李福根覃灏金其煌谢正华熊磊
Owner 3D BIOMEDICINE SCI & TECH CO LTD
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