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Construction method of fusion gene detection library based on Ion Torrent sequencing platform and fusion gene detection method

A sequencing platform and gene fusion technology, applied in the biological field, can solve the problems of inability to detect gene fusions, inability to detect unknown fusion breakpoint fusion types, etc.

Inactive Publication Date: 2017-01-04
天津诺禾医学检验所有限公司
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AI Technical Summary

Problems solved by technology

[0006] The present invention aims to provide a method for constructing a fusion gene detection library based on the Ion Torrent sequencing platform and a method for detecting fusion genes, so as to solve the problem that the Ion Torrent sequencing platform cannot detect gene fusion and cannot detect unknown genes due to unqualified RNA samples in the prior art. Fusion Type Issues for Fusion Breakpoints

Method used

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  • Construction method of fusion gene detection library based on Ion Torrent sequencing platform and fusion gene detection method
  • Construction method of fusion gene detection library based on Ion Torrent sequencing platform and fusion gene detection method
  • Construction method of fusion gene detection library based on Ion Torrent sequencing platform and fusion gene detection method

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Experimental program
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Embodiment approach

[0037] According to a typical implementation of the present invention, the method includes the following steps:

[0038] 1. Fragmentation of the DNA of the sample to be tested

[0039] Use an ultrasonic fragmentation instrument (Covaris LE220R) to fragment the sample into DNA fragments with a main band of 150bp to 300bp according to the fragmentation parameters in Table 1; especially, if the sample DNA is less than 100ng, 500ng of Carrier RNA should be added for auxiliary fragmentation.

[0040] Table 1

[0041]

[0042] 2. DNA fragment end repair and A base addition at the 3' end;

[0043] 3. Joint connection

[0044] The end of the random fragment of DNA connected to A base is connected to the adapter with known sequence (the sequence is known, and a blocking reagent for the adapter sequence can be used in the subsequent hybridization capture).

[0045] 4.PCR amplification

[0046] Perform PCR amplification based on known adapter sequence primers to obtain a sample nu...

Embodiment 1

[0054] 3 cases of FFPE fusion-positive samples from patients with non-small cell lung cancer, the sample name, fusion gene and type, extraction amount and usage are shown in Table 2.

[0055] Table 2

[0056]

[0057] 1. Fragmentation of DNA

[0058] Use a sonicator (Covaris LE220R) to fragment the target DNA and the incorporated Carrier RNA (500ng) according to specific shearing parameters. The shearing parameters of Covaris LE220R are set as shown in Table 1.

[0059] The fragmented DNA was purified by 1.8X AMPure XP magnetic beads before proceeding to the next step.

[0060] 2. End repair and A base addition at the 3' end

[0061] Refer to the ratio in Table 3 to prepare the reaction mixture, and gently blow up and down with a gun to mix well.

[0062] table 3

[0063]

[0064] Put it into the PCR machine, and carry out the reaction according to the setting program in Table 4 (the cover temperature is set to 70°C, the heat cover is not covered for the first 30 min...

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Abstract

The invention discloses a construction method of a fusion gene detection library based on an Ion Torrent sequencing platform and a fusion gene detection method. The construction method comprises the following steps that 1, fragmentation is conducted on sample DNA to obtain DNA fragments; 2, tail end repairing and basic group A adding are conducted on the DNA fragments; 3, joints with known sequences are connected; 4, PCR amplification is conducted to obtain a sample nucleic acid library; 5, the sample nucleic acid library captures DNA fragments of fusion genes and partner genes on the basis of probe hybridization of a target area capturing platform; 6, a product obtained in the step 5 is introduced into a sequencing joint sequence through PCR, and the fusion gene detection library is obtained. By means of the technical scheme, the problems that in the prior art, due to the fact that an RNA sample is not qualified, the Ion Torrent sequencing platform cannot detect the fusion genes and cannot detect the fusion types of unknown fusion breakpoints are solved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a fusion gene detection library based on the Ion Torrent sequencing platform and a method for fusion gene detection. Background technique [0002] Gene fusion is a type of gene structural variation, which refers to a chimeric gene formed by connecting the coding regions of two or more genes end to end and under the control of the same set of regulatory sequences. The expression product of the fusion gene is a fusion protein. [0003] With the continuous reduction of the cost of massively parallel sequencing (next-generation sequencing) and the continuous maturity of target region capture technology, gene detection based on next-generation sequencing technology has been more and more applied. Compared with traditional detection methods, next-generation sequencing has the advantages of high sensitivity, high accuracy, low cost, and the ability to simultaneous...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C40B50/06C12Q1/68
CPCC12N15/1093C12Q1/6806C12Q1/6869C40B50/06
Inventor 蒋智张振宇师文霞李晓敏夏贇高长欣
Owner 天津诺禾医学检验所有限公司
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