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ALK gene and EML4 gene detection probe, preparing method thereof and kit

A gene detection and detection probe technology, applied in the field of ALK gene and EML4 gene detection probes and their preparation, can solve the problems of high detection kits and lack of specificity, and achieve the effects of good repeatability and accurate signal counting

Inactive Publication Date: 2016-05-04
GUANGZHOU LBP MEDICINE SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is still a lack of specific detection kits for ALK / EML4 gene FISH detection

Method used

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  • ALK gene and EML4 gene detection probe, preparing method thereof and kit
  • ALK gene and EML4 gene detection probe, preparing method thereof and kit
  • ALK gene and EML4 gene detection probe, preparing method thereof and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The preparation of embodiment 1ALK gene and EML4 gene detection probe

[0043] The preparation method of the described ALK detection probe of this implementation comprises the following steps:

[0044] Select the clone containing the target gene ALK and both ends of the sequence, such as Figure 1A shown. GSPALK includes two sets of probes:

[0045] The first group of probes includes the first probe, the second probe and the third probe, as shown in the following table, which were purchased from Invitrogen RP11BAC and CTDBAC clone libraries.

[0046] The second group of probes includes the fourth probe, the fifth probe and the sixth probe, as shown in the following table, which were purchased from Invitrogen RP11BAC and CTDBAC clone libraries.

[0047] The following two groups of detection probes were prepared respectively.

[0048] Probe set 1—labeled red R, total length of BAC: 462Kb.

[0049] Probe set 2—labeled green G, total length of BAC: 513Kb.

[0050] ...

Embodiment 2

[0067] Example 2: ALK gene detection, kits for ALK gene and EML4 gene detection

[0068] This embodiment provides two kinds of kits, namely ALK gene detection kit, and ALK gene and EML4 gene detection kits.

[0069] The ALK gene rearrangement detection kit includes two components of a hybridization solution and a DAPI counterstain, wherein the hybridization solution contains two groups of GSPALK probes (two-color) described in Example 1, and a buffer for the hybridization environment (to promote hybridization) Liquid components, COTHumanDNA with closed repeat 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.

[0070] The specific formula is as follows:

[0071] Hybridization solution preparation

[0072]

[0073] ALK gene and EML4 gene detection kit in...

Embodiment 3

[0082] Embodiment 3: the detection method of ALK gene and EML4 gene detection kit

[0083] 1. Slide pretreatment

[0084] 1. Place the slides in a constant temperature oven at 65±5°C and bake overnight;

[0085] 2. Take out the slide and place it in xylene (or environmental dewaxing agent) at room temperature for 10 minutes;

[0086] 3. Take out the slide, and put it in another cylinder of room temperature xylene (or environmental dewaxing agent) for 10 minutes;

[0087] 4. Take out the slide, and put it in absolute ethanol at room temperature for 10 minutes to remove residual xylene (or environmental

[0088] dewaxing agent);

[0089] 5. Take out the slide, and put it into 100%, 90%, 70% graded ethanol for rehydration at room temperature for 3 minutes each;

[0090] 6. Take out the slide, put it in sterilized purified water and wash it at room temperature for 3 minutes, and absorb excess water with a lint-free paper towel;

[0091]7. Take out the slide, put the slide int...

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Abstract

The invention relates to an ALK gene detection probe, an ALK gene and EML4 gene detection probe and a preparing method thereof. The preparing method comprises the following steps that BAC aiming at an ALK gene is selected to be cloned into at least one of RP11-62B19, RP11-100C1 and CTD-2245E6, and BAC is selected to be cloned into at least one of CTD-2544I11, RP11-684O3 and RP11-134O13; BAC aiming at an EML4 gene is selected to be cloned into at least one of CTD-2358L8 and CTD-2547J15; plasmid DNA is obtained; marking is carried out. The invention further discloses a kit containing the ALK gene and EML4 gene detection probe. The optimal ALK gene and EML4 gene detection probe is obtained by means of screening, signal counting is accurate and fast, and result repeatability is good.

Description

technical field [0001] The invention belongs to biotechnology, and in particular relates to ALK gene and EML4 gene detection probes, preparation methods and kits thereof. Background technique [0002] In 2014, the annual report of tumor registration indicated that lung cancer ranked first in the incidence rate (35.23 / 100,000, 19.59%) and death rate (27.93 / 100,000, 24.87%) of malignant tumors in China. The occurrence of lung cancer is considered to be a multi-step process, which is closely related to the amplification / activation of proto-oncogenes and / or the inactivation of tumor suppressor genes. opportunity for treatment. The study found that 64% of lung adenocarcinoma patients detected cancer drivers, of which 8% were ALK rearrangements. ALK gene fusion is not a favorable prognostic factor for advanced NSCLC, but crizotinib can improve the survival of ALK-positive patients. Previous studies have shown that the EML4-ALK gene fusion mutation rate is 2% to 7%. A study rep...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12N15/1034C12Q1/6886C12Q2600/156C12Q2531/113C12N15/10C12N15/11C12Q1/68
Inventor 何瑰陈绍宇欧焕金张会清
Owner GUANGZHOU LBP MEDICINE SCI & TECH
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