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Probe set for detecting BRAF-related fusion gene, kit and application thereof

A technology of fusion genes and probe sets, applied in DNA/RNA fragments, recombinant DNA technology, determination/inspection of microorganisms, etc., can solve the problem of insufficient accuracy of gene fusion, and make it difficult to miss complex variant types, signal count lines, etc. Accurate and discriminative effect

Pending Publication Date: 2019-09-03
GUANGZHOU LBP MEDICINE SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantage of this method is that it is affected by the amount of enzyme used and the preference of enzyme digestion, and there are differences in the labeling efficiency for different templates, so that the accuracy of the labeled probes for detecting gene fusions is insufficient.

Method used

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  • Probe set for detecting BRAF-related fusion gene, kit and application thereof
  • Probe set for detecting BRAF-related fusion gene, kit and application thereof
  • Probe set for detecting BRAF-related fusion gene, kit and application thereof

Examples

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preparation example Construction

[0037] In some embodiments, the present invention provides a detection probe set of BRAF gene and its partner genes and its preparation method. The prepared probe set can be used to detect the status of BRAF-related fusion genes, and its partner genes include: KIAA1549, SRGAP3, FAM131B, AKAP9; that is, the probe set is used to detect BRAF / KIAA1549, BRAF / SRGAP3, BRAF / FAM131B, BRAF / AKAP9 fusion genes; in some embodiments, the present invention provides a BRAF-related fusion gene detection kit , to directly observe the signal in tissue samples to determine whether BRAF is abnormal and the type of fusion, with good specificity.

[0038] In some embodiments, the probe set of the present invention includes 4 sets of dual-color probes for detecting BRAF / KIAA1549, BRAF / SRGAP3, BRAF / FAM131B, BRAF / AKAP9 fusion genes, and the design process includes: BAC clone selection → purchase (commercialization)→marking, testing→confirmation of the probe clone set (available for subsequent productio...

Embodiment 1

[0054] Embodiment 1 Probe set design and preparation

[0055] (1) Probe design

[0056] The probe set used to detect BRAF-related fusion genes in this example includes 4 sets of dual-color probes, involving BRAF and partner genes KIAA1549, SRGAP3, FAM131B, and AKAP9. The design process includes: BAC clone selection → purchase (commercialization) → labeling , Test→Confirm clone group (can be used for subsequent production).

[0057] The fusion gene probe design process involves two genes fused. In this embodiment, the relevant genes that are fused with the BRAF gene are mainly detected. Therefore, the first gene is the BRAF gene, and the probe set including the BRAF gene is designed for the BRAF gene. Rearrangement / break detection; the second gene is the partner gene of BRAF fusion, including KIAA1549, SRGAP3, FAM131B, AKAP9. The markers of the first gene and the second gene have different colors, and red and green adjacent or overlapping fusion signals will be detected when ...

Embodiment 2

[0087] Embodiment 2 probe labeling system parameter optimization

[0088] (1) Optimization of the mixture ratio of fluorescein-dCTP and fluorescein-dUTP

[0089] In order to improve the efficiency of fluorescein incorporation, in addition to using conventional modified dUTP, the incorporation effect of different ratios of other fluorescein-dNTPs (see Table 6, where the plasmid selects the BRAF gene probe set) was explored, and the results (see Table 7) It indicated that the mixture of fluorescein-dCTP and fluorescein-dUTP (ratio 1:2~1:3) had the best labeling effect, bright fluorescent signal and good specificity.

[0090] Table 6 Solution I

[0091]

[0092] The random hexamer primer contains a random sequence of 6 bases, with a phosphate group at the 5` end and a hydroxyl group at the 3` end, the sequence is 5`-P-d(NNNNNN)-3`, N=G\A\C\T . There are many combinations of random primers, so the specificity is low, but it has a better effect on the amplification of long te...

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Abstract

The invention discloses a probe set for detecting BRAF-related fusion gene. The probe set comprises probe pairs for detecting BRAF / KIAA1549, BRAF / SRGAP3, BRAF / FAM131B, and BRAF / AKAP9 fusion genes, oneprobe in the probe pair targets the BRAF gene, and the other probe targets one of the KIAA1549, SRGAP3, FAM131B, and AKAP9 genes, and each probe in the probe set is probe-labeled with phi29 DNA polymerase. The invention also discloses a kit for detecting the BRAF-related fusion gene, which comprises the above probe set and a hybridization buffer solution, wherein the hybridization buffer solutioncontains dimethyl sulfoxide. The optimal BRAF gene and related partner gene fusion gene detection probes and the combination are screened, and a FISH method is employed for fusion gene detection, andthe signal counting is accurate and rapid. The result is reproducible; in the present invention, the phi29 DNA polymerase is used for probe labeling for the first time, and a random primer labeling method for phi29 DNA is established; and combined with fine component adjustment, the marking effect is superior to a commonly used nick translation method.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a probe set, a kit and an application thereof for detecting BRAF-related fusion genes. Background technique [0002] Malignant primary brain tumors account for about 2% of malignant tumors, and the peak age of onset is 5-10 years old and 50-55 years old. Astrocytomas are highly aggressive, and according to WHO regulations, they are graded according to the degree of tumor differentiation. Fibrocytic astrocytomas (grade I) and Pilocytic astrocytoma (WHO grade I) is less invasive, has limited manifestations, and is less likely to progress to the anaplastic phase, and is more common in children and adolescents. [0003] BRAF abnormalities are often present in low-grade central nervous system tumors, and the abnormalities involve random duplications and fusions of the BRAF gene in the 7q34 region. Studies suggest that the BRAF gene abnormally activates the MAPK pathway, whic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6841C12Q1/6886C12N15/11
CPCC12Q1/6841C12Q1/6886C12Q2600/112C12Q2527/149C12Q2527/125
Inventor 陈绍宇何瑰
Owner GUANGZHOU LBP MEDICINE SCI & TECH
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