TOP2A gene detection probe, preparation method thereof and reagent kit

A gene detection and kit technology, applied in the field of TOP2A gene detection probes and its preparation, can solve the problems of high detection kits and lack of specificity, and achieve the effects of reducing hybridization background, high sensitivity, and accurate signal counting lines

Inactive Publication Date: 2016-04-13
GUANGZHOU LBP MEDICINE SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, for the detection of TOP2A gene FISH method, there is still a lack of highly specific detection kits

Method used

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  • TOP2A gene detection probe, preparation method thereof and reagent kit
  • TOP2A gene detection probe, preparation method thereof and reagent kit
  • TOP2A gene detection probe, preparation method thereof and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The preparation of embodiment 1TOP2A gene detection probe

[0038] The preparation method of the TOP2A detection probe described in this implementation includes the following steps.

[0039] Select the clones containing the target gene TOP2A and the sequences at both ends, such as figure 1 shown.

[0040] GSPTOP2A includes two groups, respectively including the first probe, the second probe, the third probe and the fourth probe. The details are shown in the following table, which were purchased from Invitrogen RP11BAC and CTDBAC clone libraries. The following two groups of detection probes were prepared respectively.

[0041] TOP2Achr17:38,544,773-38,574,202,29,430bp

[0042] Probe set 1

BAC

Insert segment start and end position

first probe

RP11-1152A10

chr17: 38376940...38546337 (169Kb)

second probe

CTD-3217C5

chr17: 38521566...38730400 (209Kb)

[0043] Probe set 2

BAC

Insert segment start and end...

Embodiment 2

[0060] Embodiment 2: Preparation method of TOP2A gene detection kit

[0061] The TOP2A gene detection kit includes two components of TOP2A hybridization solution and DAPI counterstaining agent, wherein the TOP2A hybridization solution contains the GSPTOP2A gene probe described in Example 1 (respectively two groups of detection probes, corresponding to two kits) , CSP17 probe (identification probe for chromosome 17), buffer components for hybridization environment (promoting hybridization), COT Human DNA for blocking repetitive sequences, etc. DAPI counterstaining agent is mainly used for cell counterstaining after hybridization, in which DAPI will bind to DNA, making the nucleus show blue fluorescence, and the counterstaining agent containing p-phenylenediamine can keep the fluorescence stable.

[0062] The specific formula is as follows:

[0063] (1) Preparation of hybridization solution

[0064]

[0065] (2) DAPI counterstain preparation

[0066] Dissolve 10 mg of p-ph...

Embodiment 3

[0069] Embodiment 3: the detection method of TOP2A gene detection kit

[0070] 1. Slide pretreatment

[0071] 1.1 Place the slides in a 65±5°C incubator and bake overnight;

[0072] 1.2 Take out the slide and put it in xylene to dewax at room temperature for 15 minutes;

[0073] 1.3 Take out the slide, and put it into another vat of xylene to continue dewaxing at room temperature for 15 minutes;

[0074] 1.4 Take out the slide, and put it in absolute ethanol at room temperature for 10 minutes to remove residual xylene;

[0075] 1.5 Take out the slide, and put it into 100%, 90%, 70% graded ethanol for rehydration at room temperature for 3 minutes each;

[0076] 1.6 Take out the slide, put it in purified water and wash it at room temperature for 3 minutes, and absorb excess water with a lint-free paper towel;

[0077] 1.7 Take out the slides, put them into purified water and boil them at 100±5°C for 25 minutes (place the slides horizontally in the container with the sample s...

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Abstract

The invention relates to a TOP2A gene detection probe and a preparation method thereof. The method includes the following steps that a selected BAC clone is at least one of RP11-1152A10 and CTD-3217C5, or a selected BAC clone is at least one of RP11-737D6, CTD-3087O22, RP11-48O10 and CTD-2134K5, a plasmid is extracted from the clone to obtain DNA of the plasmid, and quantification is conducted; marking is carried out with fluorescein. The invention further discloses a reagent kit provided with the TOP2A gene detection probe. By obtaining the optimal TOP2A detection probe through screening, signal line counting is accurate and quick, and result repeatability is good. Defects of TOP2A mutation detection in clinic are made up for, more patients benefited from targeted drug can be screened more easily, the survival rate of the patients is increased, and overall life time is prolonged.

Description

technical field [0001] The invention belongs to biotechnology, and in particular relates to a TOP2A gene detection probe, a preparation method and a kit thereof. Background technique [0002] The TOP2A gene is the coding gene of DNA topoisomerase IIα (TopoisomeraseIIalpha, TOPIIα). Thereby indirectly affecting the metabolic process of nucleic acid in cells; the second is to directly participate in the cellular processes that need to break and reconnect DNA molecular chains, DNA recombination, repair, transcription and replication. The prognosis of patients with TOP2A gene abnormality is poor, and the recurrence-free survival period is shortened, especially the prognosis of patients with TOP2A gene deletion is even worse. Gene amplification indicates that the tumor may recur, or the long-term curative effect will decline. [0003] Anthracyclines are the cornerstone of postoperative adjuvant chemotherapy for breast cancer. Studies have confirmed that the TOP2A gene is relate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12N15/11C12Q1/68C12Q1/6886C12Q2600/106C12Q2600/166
Inventor 陈绍宇何瑰席影
Owner GUANGZHOU LBP MEDICINE SCI & TECH
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