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Probe set and kit for detecting ALK fusion breaking point

A kit and probe set technology, applied in the field of gene detection and molecular genetics, can solve problems such as detection difficulties, and achieve the effects of improving detection specificity, low cost and high throughput

Inactive Publication Date: 2016-06-15
北京迈基诺基因科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the ALK fusion gene can be broken at multiple sites, and at the same time it can be fused with a variety of genes, and can be divided into many types according to the different break points, which brings great difficulties to detection

Method used

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  • Probe set and kit for detecting ALK fusion breaking point
  • Probe set and kit for detecting ALK fusion breaking point
  • Probe set and kit for detecting ALK fusion breaking point

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: Design and preparation of the probe set of the present invention

[0020] The position of the whole exon sequence of the ALK gene is shown in Table 1. According to the whole exon sequence of the ALK gene, a 78bp probe sequence was designed for the non-repeated region in each region, and each sequence was designed by moving along the gene position. Using in situ synthesis technology, a large number of designed probes (MyGenostics.US) were synthesized, and a large number of biomarked probes were amplified by PCR.

[0021] Table 1:

[0022]

[0023]

[0024] Described PCR method is specifically:

[0025] Mix the probes synthesized above evenly in a total volume of 1.2 ml of dH 2 In O, take 15 μl of them and use general PCR primers (the sequence at the 5' end is GACTACATGGGACAT, and the sequence at the 3' end is GGAACCTACGACGTA), and divide into three tubes for PCR amplification, wherein the primer GACTACATGGGACAT is a primer labeled with biotin.

[0...

Embodiment 2

[0029] Embodiment 2: Composition, preparation and use of the kit of the present invention

[0030] The ALK fusion fragment sequence detection kit described in this example is a kit for molecular genetic detection by detecting mutations in the entire ALK genome.

[0031] The components contained in the kit are: the probe set obtained in Example 1, buffer HY, buffer BL, library enrichment binding buffer, washing buffer WB1, washing buffer WB2, buffer NE, PCR reaction Liquid, product purification eluent. The specific composition is:

[0032]

[0033] The using method of described test kit is:

[0034] 1. Sample library preparation:

[0035] (1) Ultrasonic fragmentation: the initial amount is 3 μg, diluted to 30 ng / μL with 1×lowTEBuffer. CovarisS2 ultrasonic instrument was used for ultrasonic fragmentation, and the value of Covaris system was set according to the standard, 6 cycles×60s, water bath temperature: 5°C, duty cycle: 20%, intensity: 5, mode: Frequencysweeping.

...

Embodiment 3

[0079] Embodiment 3: verification of the use effect of the kit of the present invention

[0080] The present invention will be further described in detail through specific embodiments below.

[0081] 1. High-throughput sequencing detection of a case of ALK fusion gene

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Abstract

The invention provides a probe set and a kit for detecting an ALK fusion breaking point. ALK whole exon sequence can be covered by a probe, and probe sequence is shown in the SEQ ID No:1-230. Based on the gene trapping sequencing technique, the method for finding out the breaking point through whole exon depth trapping and high-throughput sequencing of the ALK fusion gene is provided, and individual primer prognosis is designed for the breaking point for monitoring. The invention further provides the kit for detecting the ALK fusion breaking point. The kit comprises a trapping probe capable of detecting the ALK fusion breaking point and relevant reagents. By the adoption of the probe set and the kit, ALK fusion sequence can be quickly and accurately detected.

Description

technical field [0001] The invention belongs to the fields of gene detection and molecular genetics, and in particular relates to a probe group and a kit for detecting ALK fusion breakpoints. Background technique [0002] ALK fusion gene is an important basis for patients with non-small cell lung cancer (NSCLC) to receive targeted therapy. Although ALK-positive NSCLC only accounts for 5% of all NSCLC, the number of new cases in China is still close to 30,000 each year. At the same time, the ALK fusion gene can be broken at multiple sites, and at the same time it can be fused with a variety of genes, and can be divided into many types according to the different break points, which brings great difficulties to detection. In order to accurately detect the ALK fusion gene and use gene capture plus next-generation sequencing technology to detect the ALK fusion gene. In summary, the present invention provides a capture probe set and kit for detecting ALK fusion. Contents of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6886C12Q2600/118C12Q2535/122C12Q2565/519
Inventor 伍建林朋
Owner 北京迈基诺基因科技股份有限公司
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