A method and kit for constructing alk gene fusion mutation detection library

Abstract

The invention provides a method for constructing and detecting ALK gene fusion mutation library comprising the following steps: 1) extracting DNA and fragmenting it; 2) constructing a DNA pre-library; 3) circularizing DNA; 4) using ALK specific PCR amplification was performed with specific primers, and the target region was enriched to generate the final library. The invention also provides a kit for constructing a library for detecting ALK gene fusion mutations and a method for detecting ALK gene fusion mutations. The library construction method targeted at ALK recombination hotspots provided by the present invention combined with the second-generation high-throughput sequencing technology has excellent sensitivity, and can accurately detect information on ALK gene fusion mutations using plasma DNA or tissue DNA as starting materials. In addition, when plasma DNA is used as the starting material, the present invention also realizes the non-invasive detection of ALK gene fusion mutation and crizotinib resistance mutation.

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-SmallCell 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 developmen...

Examples

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 XPbeads 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 ...

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-SmallCell 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 developmen...

Examples

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 XPbeads 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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