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Method and kit for building ALK gene fusion mutation detection library

A gene fusion, mutation library technology, applied in chemical libraries, biochemical equipment and methods, combinatorial chemistry, etc., can solve the problems of blank, cancer cell metastasis, and the inability to track the progress of patients' disease.

Active Publication Date: 2016-11-23
BERRY ONCOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This invasive detection method is accompanied by unnecessary suffering of patients and the risk of cancer cell metastasis
And biopsy may bring false negative results due to the heterogeneity of the sampling site
Invasive tissue sampling is also not reproducible, and patient progress cannot be tracked
In terms of non-invasive detection of ALK gene fusion mutations, the status quo is still blank

Method used

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  • Method and kit for building ALK gene fusion mutation detection library
  • Method and kit for building ALK gene fusion mutation detection library
  • Method and kit for building ALK gene fusion mutation detection library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] The method for constructing an ALK fusion gene mutation detection library using plasma DNA as a starting material mainly includes the following steps:

[0082] (1) Extraction of plasma DNA: This step can be carried out by using any method and reagent suitable for extracting plasma DNA well known to those skilled in the art.

[0083] (2) End filling, followed by cleaning and purification of the product: this step can be performed using any methods and reagents known to those skilled in the art that are suitable for end filling and subsequent cleaning and purification. For example, T4 DNA polymerase, Klenow enzyme can be used to fill in blunt ends.

[0084] (3) Overhang A at the end, followed by cleaning and purification of the product: This step can be performed using any methods and reagents known to those skilled in the art that are suitable for overhang A at the end and subsequent cleaning and purification of the product. For example, under the action of klenow ex-(N...

Embodiment 2

[0124]The method of constructing plasma DNA detection ALK gene fusion mutation library in Example 2 is basically similar to Example 1, the difference is that: in Example 2, the end filling and the end hanging A are carried out in one reaction system, saving the intermediate Purification steps. A specific example of constructing an ALK gene fusion mutation detection library using the method according to Example 2 of the present invention is shown below.

[0125] Step 1: Extract about 20ng of plasma DNA.

[0126] Step 2: Prepare the reaction mixture of filling in the end and hanging A at the 3' end as shown in Table 10, incubate at 37°C for 20 minutes, and incubate at 72°C for 20 minutes, so as to perform end filling and end in one reaction system Suspension A; DNA samples were purified on a purification column, and in 37 μl of sterile dHO 2 O or elution buffer. According to the composition of different reaction mixtures suitable for different design needs, the temperature an...

Embodiment 3

[0132] A specific example of sequencing a tissue genome DNA sample using the method according to the present invention is shown below.

[0133] Step 1: Extract about 200ng of tissue DNA and dissolve it in 25μl EB.

[0134] Step 2: Prepare the reaction mixture shown in Table 11, fragment the tissue DNA to about 200 bp with fragmentase, react at 37°C for 20 minutes, add 5 μl of 0.5M EDTA to terminate the reaction after the reaction is completed. DNA fragments interrupted with Beckman Ampure XP beads and washed with 41.5 μl of sterile dH 2 O or elution buffer.

[0135] Table 11

[0136]

[0137] Step 3: Prepare the reaction mixture of end filling and 3' suspension A as shown in Table 12, incubate at 37°C for 20 minutes, and incubate at 72°C for 20 minutes, so as to perform end filling and end suspension in one reaction system out; DNA samples were purified on a purification column, and in 37 μl of sterile dH 2 O or elution buffer. According to the composition of different...

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Abstract

The invention provides a method for building an ALK gene fusion mutation detection library. The method includes the steps of firstly, extracting DNA, and fragmenting the DNA; secondly, building a DNA pre-library; thirdly, cyclizing the DNA; using ALK specific primers to perform PCR amplification, and enriching a target area to generate the final library. The invention further provides a kit for building the ALK gene fusion mutation detection library and a method for detecting ALK gene fusion mutation. The method for building the ALK gene fusion mutation detection library has the advantages that the method targeted at ALK recombination hot spot regions is excellent in sensitivity when being combined with the second-generation high-through sequencing technology, and plasma DNA or tissue DNA can be used as the initial material to accurately detect the information of ALK gene fusion mutation; in addition, when the plasma DNA is used as the initial material, noninvasive detection of ALK gene fusion mutation and crizotinib drug-resistant mutation is achieved.

Description

technical field [0001] The invention relates to a method and a kit for constructing an ALK fusion gene mutation detection library. The invention also relates to a method for realizing high-sensitivity and accurate detection of ALK gene fusion mutation by performing high-throughput sequencing on the ALK fusion gene mutation detection library. Background technique [0002] Anaplastic lymphoma kinase (ALK) is a tyrosine receptor kinase composed of an extracellular domain, a transmembrane domain and an intracellular kinase domain. In 1%-7% of non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) patients, fusion of mutated ALK gene and other genes was found, resulting in high expression activity of intracellular tyrosine kinase. The genes that have been found to be fused with the ALK gene include EML4, KIF5B, TGF, KLC1, PTPN3, STRN, etc., and the fusion sites are diverse. There are more than 20 known fusion sites between EML4 and ALK. With the deepening and developme...

Claims

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Application Information

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IPC IPC(8): C12N15/10C40B50/06C12Q1/68
Inventor 张亚晰茹兰兰王丽娜张建光
Owner BERRY ONCOLOGY CO LTD
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