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A fluorescent in situ hybridization probe for detecting kiaa1549-braf fusion gene and its preparation method and application

Active Publication Date: 2019-08-09
WUHAN HEALTHCHART BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the design region of the existing BRAF breakage probe is 550Kb around the BRAF gene breakage area, when the BRAF gene breaks, the distance between the signal points is 1.4Mb, which is relatively short and cannot be distinguished from the breakage signal points; and KIAA1549 / BRAF fusion gene probe design region ( figure 2 ) fusion regions of the two genes respectively, and one more fusion signal appeared in the middle of the two signal points after the fusion, but the distance between the three signal points was about 1.0Mb, and the break signal points could not be distinguished as well.
Therefore, neither the conventional BRAF breakage probe nor the KIAA1549 / BRAF fusion gene probe can fully distinguish positive and negative samples at present

Method used

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  • A fluorescent in situ hybridization probe for detecting kiaa1549-braf fusion gene and its preparation method and application
  • A fluorescent in situ hybridization probe for detecting kiaa1549-braf fusion gene and its preparation method and application
  • A fluorescent in situ hybridization probe for detecting kiaa1549-braf fusion gene and its preparation method and application

Examples

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Embodiment 1

[0045] Example 1 Preparation of KIAA1549-BRAF fusion gene rapid detection probe

[0046] The preparation of the KIAA1549-BRAF fusion gene rapid detection probe of the present invention comprises the following steps:

[0047] (1) Download the genome non-repeating sequence of the region covered by the KIAA1549 and BRAF gene probes from the UCSC Genome Browser, where the KIAA1549 probe covers the region: chr7 138,361,140-138,861,139 (the starting position is exon 16 of the KIAA1549 gene toward the centromere 500Kb region); BRAF gene probe coverage area is: chr7 140,787,547-141,287,546 (the starting position is the 500kb region from exon 9 of BRAF gene toward the telomere); the obtained sequence is saved in fasta format, and the repetitive sequence region in the genome is replaced by N;

[0048] (2) Use the perl plug-in program chunks.pl to segment the non-repeated sequence of the genome in the area covered by the KIAA1549 gene probe obtained in step (1) into blocks of 1 kb size;...

Embodiment 2

[0055] Example 2 The method of using the rapid detection probe of KIAA1549-BRAF fusion gene

[0056] The method for using the KIAA1549-BRAF fusion gene rapid detection probe prepared in Example 1 when detecting the KIAA1549-BRAF fusion gene specifically includes the following steps:

[0057] (1) Sample treatment: Take a paraffin section sample with a thickness of 4-5 microns that has been baked, and immerse it in preheated 65°C dewaxing agent I for 10-15 minutes; take out the slide and immerse it in a preheated 65°C dewax immerse in 100% ethanol, 85% ethanol, and 70% ethanol at room temperature for 2 to 3 minutes; take out the slide and immerse in preheated 65°C deionized water for 3 to 3 minutes. 5 minutes; take out the slide and immerse in 95°C deionized water for 30-40 minutes, pre-cool the slide in cold water for 1 minute; take out the slide and immerse in the preheated 37°C protease working solution for 20-40 minutes; take out the slide and soak in Rinse twice in the elu...

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Abstract

The invention belongs to the field of molecular biology, in particular to a fluorescence in situ hybridization probe for detecting KIAA1549-BRAF fusion gene and preparation method and application thereof. The fluorescence in situ hybridization probe comprises two probe libraries, a KIAA1549 gene hybridization probe library: the starting position of the probe is designed in the range of 200 Kb around the KIAA1549 gene breakpoint, the probe extends toward the direction of the centromere, and the length of the probe is 300 to 1000 Kb; BRAF gene hybridization probe library: The starting position of the probe is designed in the range of 200 Kb around the breakpoint of BRAF gene, and the probe extends to the telomere. The length of the probe is 300 - 1000 Kb. The invention designs probes in twofusion gene outer regions respectively, realizes the detection of two fusion genes which are relatively close on the same chromosome, and can directly reflect the fusion state of the two genes on thecell level.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a fluorescent in situ hybridization probe for detecting KIAA1549-BRAF fusion gene, a preparation method and application thereof. Background technique [0002] Pilocytic astrocytoma (PA) is a primary intracranial tumor classified as grade I by WHO. It is the most common glioma in children's neurosurgery, accounting for about 25% of brain tumors. Both the KIAA1549 gene and the BRAF gene are located in the q34 region of chromosome 7. The tandem duplication of the BRAF gene leads to the KIAA1549 / BRAF gene fusion, which is highly common in pilocytic astrocytomas (60%-80%). According to the different fusion positions of KIAA1549 and BRAF genes, five different fusion variants have been reported, namely: KIAA1549ex16-BRAF ex9, KIAA1549ex15-BRAF ex9, and KIAA1549ex16-BRAF ex11, KIAA1549ex18-BRAF ex10 and KIAA1549ex19-BRAF ex9; The three kinds accounted for 45%, 28% and 5% of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6841C12N15/11
CPCC12Q1/6841C12Q1/6886C12Q2563/107
Inventor 吕玉琦李先洋李倩李雪梅叶伦程弘夏陈刚
Owner WUHAN HEALTHCHART BIOLOGICAL TECH