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AML1 gene and ETO gene detection probe, preparation method thereof and reagent kit

A gene detection and gene technology, applied in the field of AML1 gene and ETO gene detection probes and their preparation, can solve the problems of lack of specificity, high detection kits, etc., achieve good discrimination, improve survival rate, and good specificity Effect

Pending 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, there is still a lack of highly specific detection kits for AML1 / ETO gene FISH detection

Method used

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  • AML1 gene and ETO gene detection probe, preparation method thereof and reagent kit
  • AML1 gene and ETO gene detection probe, preparation method thereof and reagent kit
  • AML1 gene and ETO gene detection probe, preparation method thereof and reagent kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The preparation of embodiment 1AML1 gene and ETO gene detection probe

[0037] The preparation method of the AML1 / ETO detection probe described in this implementation comprises the following steps:

[0038] Select the clones containing the target genes AML1 and ETO and the sequences at both ends, as shown in Figures 1 and 2.

[0039] GSPAML1 includes the first probe, the second probe, the third probe, the fourth probe and the fifth probe, as shown in the table below, which were purchased from Invitrogen RP11BAC and CTDBAC clone libraries.

[0040] GSPETO includes the first probe, the second probe, the third probe, the fourth probe and the fifth probe, as shown in the table below, which were purchased from Invitrogen RP11BAC and CTDBAC clone libraries.

[0041] The following two groups of detection probes were prepared respectively.

[0042] AML1

[0043]

[0044]

[0045] ETO

[0046]

[0047] (2) Preparation of gene probes: using Qiagen’s PlasmidMaxiKit, e...

Embodiment 2

[0058] Embodiment 2: AML1 and ETO gene detection kit preparation method

[0059] AML1 and ETO gene detection kit include two components of AML1 and ETO hybridization liquid and DAPI counterstaining agent, wherein AML1 and ETO hybridization liquid comprise GSPAML1 and GSPETO gene probe described in embodiment 1, are used for hybridization environment (promoting Hybridization) buffer components, COT Human DNA 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.

[0060] The specific formula is as follows:

[0061] Hybridization solution preparation

[0062]

[0063] a. DAPI counterstain preparation

[0064] Dissolve 10 mg of p-phenylenediamine in 1 ml of PBS, adjust the pH to 9.0, add 9 ml of glycerin, shake and mix repeatedly, and store ...

Embodiment 3

[0067] Embodiment 3: the detection method of AML1 / ETO gene detection kit

[0068] 1. Sample processing

[0069] 1.1 Take 2-3ml of peripheral blood or bone marrow (anticoagulated with sodium heparin) and centrifuge at 2000rpm for 5min, carefully remove the supernatant.

[0070] 1.2 Add 10ml of hypotonic solution (0.075mol / LKCl), mix by pipetting, and let stand for 3min.

[0071] 1.337 ± 1 ℃ water bath box hypotonic 30min.

[0072] 1.4 Add 1ml of fresh fixative, mix by pipetting, and pre-fix at room temperature for 10min.

[0073] 1.5 Mix by pipetting and centrifuge at 2000rpm for 5min.

[0074] 1.6 Remove the supernatant, add 5-10ml of fresh fixative to the sediment, mix by pipetting, and let stand at room temperature for 10min.

[0075] 1. Centrifuge at 72000rpm for 5min, remove the supernatant.

[0076] 1.8 The above washing steps can be repeated until the cell pellet is washed white (this step does not need to stand at room temperature for 10 minutes).

[0077] 2. Prod...

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Abstract

The invention relates to an AML1 gene and ETO gene detection probe and a preparation method thereof. The method includes the following steps that a selected BAC clone for an AML1 gene is at least one of CTD-3245J1, RP11-77G18, CTD-2349F18, CTD-3171K21 and RP11-384N13, a selected BAC clone for an ETO gene is at least one of RP11-1107H4, RP11-302P1, CTD-2547C5, RP11-55J5 and CTD-2079N17, the DNA of a plasmid is obtained, and marking is conducted. The invention further discloses a reagent kit provided with the AML1 gene and ETO gene detection probe. By obtaining the optimal AML1 gene and ETO detection probe through screening, signal line counting is accurate and quick, and result repeatability is good.

Description

technical field [0001] The invention belongs to biotechnology, and in particular relates to AML1 gene and ETO gene detection probes, preparation methods and kits thereof. Background technique [0002] Acute myeloid leukemia t(8;21)(q22;q22)(AML1 / ETO) is a type of AML that usually manifests as neutrophils with mature differentiation and is one of the most common chromosomal structural abnormalities in AML, accounting for It accounts for 1 / 3 of AML with abnormal karyotype and maturity. The fusion gene can be seen in about 8% of adults and 12% of children with acute myeloid leukemia, among which AMLM2 (50%) and AMLM2b (90%) are more common. The t(8;21)(q22;q22) translocation affects the AML1 gene (also known as RUNX1), which encodes the CBFa and ETO genes. Gene transcripts were detectable in AML with t(8;21) patients. Breaks in the AML1 gene occur within a single intron. [0003] AML1 / ETO fusion is considered a cytological and molecular genetic abnormality with good prognos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12N15/10C12N15/11C12Q1/68C12Q1/6886C12Q1/6811C12Q1/6841C12Q2563/107
Inventor 陈绍宇何瑰张会清
Owner GUANGZHOU LBP MEDICINE SCI & TECH
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