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Method and kit for organism fusion gene detection and fusion abundance quantification

A technology of fusion genes and organisms, which can be used in biochemical equipment and methods, and the determination/inspection of microorganisms. , the effect of reducing the risk of opening tubes / pipetting

Inactive Publication Date: 2021-05-14
上海睿璟生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection kits based on the next-generation sequencing platform on the market are commonly used to build libraries based on the probe capture method at the DNA level, but the cost is high and the operation process is cumbersome; while reverse transcription at the RNA level plus multiple PCR technology combines the sensitivity of qPCR and specificity, as well as the intuitiveness of NGS technology results, easy operation, short time-consuming, only one sample can detect all fusion types, and has outstanding methodological advantages
[0004] However, in many existing technical solutions, most of them reverse the RNA to cDNA first, and then use the cDNA to construct the library (patent numbers: CN111088365A, CN110241215A, CN109371139A). These methods not only increase the experimental operation process, but also increase the risk of contamination. risk, and its detection of fusion gene information is relatively small, and its clinical use is limited
The current existing technology (patent number: CN 104894271A) defines the fusion positive threshold based on the value of Reads. This method is extremely unstable and is easily affected by various factors such as the amount of sequencing data and methodology.

Method used

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  • Method and kit for organism fusion gene detection and fusion abundance quantification
  • Method and kit for organism fusion gene detection and fusion abundance quantification
  • Method and kit for organism fusion gene detection and fusion abundance quantification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 The primer sequence for amplifying tumor-related gene mutation sites was synthesized by Shanghai Sangong, a primer synthesis company.

[0046] Taking CCDC6-RET as an example, the RNA primer design principle of the present invention will be described in detail below. Such as figure 1 As shown, with the position of the breakpoint as the boundary, exon 1 of the 5' end CCDC6 gene is fused with exon 12 of the 3' RET gene, so the upstream primer is designed on exon 1 of the CCDC6 gene, and the downstream primer is designed on the RET gene On exon 12 of the gene; the RET wild-type upstream primer was designed on exon 11, and the downstream was consistent with the downstream primer of the fusion gene.

[0047] (1) RNA-specific primers, the primer sequences of which are shown in Table 2:

[0048] Table 2 fusion gene and its primer sequence

[0049]

[0050]

[0051] Wild-type gene and its primer sequence

[0052]

[0053]

[0054] *P stands for phosphor...

Embodiment 2

[0061] Example 2: Sample pretreatment and nucleic acid extraction

[0062] Qualified professionals take samples with a puncture needle, and the samples include puncture tissue samples greater than 1 mg. After the biopsy is completed, infiltrate the tissue completely in RNA preservation solution as soon as possible. Samples were stored at -20°C prior to sample shipment.

[0063] Use the tissue cell RNA extraction kit (spin column method) and refer to the instructions of the kit to extract the RNA in the sample.

[0064] 1. Mechanical tissue sample homogenization:

[0065] Put the sample in a suitable glass tube or centrifuge tube, add 350 μL Buffer RL and DTT mixed lysate (100:1) lysate, insert the probe into the lysate, and homogenize intermittently at high speed, each time for 15-20 seconds until the sample Homogenize completely.

[0066] 2. Extraction of nucleic acid after homogenization

[0067] About 350 μL of the homogenized sample was centrifuged at 14000 rpm for 3 ...

Embodiment 3

[0078] Embodiment 3: Construction of library

[0079] The library construction process of this kit is as follows:

[0080] (1) RNA reverse transcription and the first round of PCR amplification (amplification of the target region) uses RNA as a template, and the reverse primer of the fusion 3' end gene with 6 random bases is used as a specific primer for reverse transcription to obtain cDNA ; Use specific multiple PCR primers and amplification reagents to amplify cDNA to obtain specific target fragments, and the reaction is carried out in a single tube. Vazyme's reverse transcriptase and its KAPA multi-reconstruction library Mix were used to prepare the following system:

[0081]

[0082] Set the following conditions on the PCR instrument for the reaction:

[0083]

[0084] (2) The first round of PCR product purification

[0085] 1) After the programmed reaction, the sample was briefly centrifuged, and the sample was added with nucleic acid-free water to make up 20 μL, ...

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Abstract

The invention discloses a method and a kit for organism fusion gene detection and fusion abundance quantification in the technical field of molecular biology. The kit is used for quantitatively detecting the abundance of various variation forms of a fusion gene in an organism by combining a multiplex PCR capture technology with a molecular tag deduplication technology; based on the detection result and a clinical pathology analysis result, the kit can provide help for scientific research, new drug development or auxiliary diagnosis and treatment of tumors.

Description

technical field [0001] The invention relates to a method and a kit for detection of organism fusion genes and quantification of fusion abundance in the field of molecular biology technology. Background technique [0002] Gene fusion (Gene Fusion) refers to connecting the coding regions of two or more genes end to end and placing them under the control of the same set of regulatory sequences (including promoters, enhancers, terminators, etc.) to form a chimeric gene, often In the way of breaking itself and joining with another gene, the 3' end gene containing the kinase domain coding region is fused with the 5' end of various heterologous upstream partner genes, and a new gene (fusion gene) is recombined. Gene fusions usually result from chromosomal rearrangements. Aberrant gene fusion events are associated with tumor development, so changes in these genes are important diagnostic, prognostic, and predictive biomarkers. [0003] For fusion detection, the current clinical go...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686
CPCC12Q1/6886C12Q1/686C12Q2600/156C12Q2537/143
Inventor 王宝霞胡春旭何文天高伙妮
Owner 上海睿璟生物科技有限公司
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