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Probe kit for detecting target gene copy-number alteration in t(8;21) AML

A gene probe and kit technology, applied in the field of t(8 detection, can solve the problems of low resolution, few counted cells, and inability to analyze the coexistence of multiple gene abnormalities, and achieve high signal strength and stability. strong effect

Active Publication Date: 2014-03-12
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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Problems solved by technology

However, the clinical indicators currently used are too low in the detection of tumor cell heterogeneity.
Take chromosome karyotype analysis as an example: Although it can detect the juxtaposition of multiple genomic abnormalities at the single-cell level, the resolution is too low (about 10Mb), and it can only detect large fragment deletions, amplifications, and heterolocations, etc., and Chromosomal karyotype analysis can only detect cells in the metaphase of mitosis, and the number of counted cells is small, which can only reflect the genetic changes of actively proliferating cells. Although the sequencing technology has a very high resolution, it can obtain a large amount of genomic abnormal information and can also observe the advantages The evolution of mutations, but it can only reflect the existence and evolution of heterogeneity at the overall level, and cannot analyze the simultaneous existence of multiple genetic abnormalities at the same cell level

Method used

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  • Probe kit for detecting target gene copy-number alteration in t(8;21) AML
  • Probe kit for detecting target gene copy-number alteration in t(8;21) AML
  • Probe kit for detecting target gene copy-number alteration in t(8;21) AML

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: Preparation of AML1 gene probe

[0040] 1. Acquisition of probe DNA

[0041] 1) Streak inoculation: Three BAC strains carrying the AML1 gene (RP11-77G18, RP11-697C13, RP11-177L11; purchased from Children's Hospital Oakland Research Institute) were operated according to the following steps. First, the BAC strains Streak inoculation on the agar plate containing antibiotic (chloramphenicol), incubate overnight (about 14 hours) at 37°C.

[0042] 2) Initial culture: Pick a monoclonal strain with good growth the next day, inoculate it in 2ml of LB liquid medium containing antibiotics (chloramphenicol), put it in a shaker at 37°C, set the shaker speed to about 200rpm, and culture 6-8 hours.

[0043] 3) Overnight culture: Transfer each 1ml of the bacterial solution obtained in step 2) to 200ml of LB liquid medium containing antibiotics (chloramphenicol), place it on a shaker at 37°C with a rotation speed of 200rpm, and cultivate overnight (about 14-16 hours).

...

Embodiment 2

[0088] Example 2: Preparation of Green-ETO (green), PF555–AML1 (yellow), PF590-WT1 (red), HyPer5-p27 (purple), PF415-c-kit (blue) probe combinations

[0089] 1. Acquisition of probe DNA

[0090] 1) Streak inoculation: will carry AML1 (RP11-77G18, RP11-697C13, RP11-177L11), ETO (RP11-118O8, RP11-122C21), WT1 (RP11-258G7, RP11-955L20), p27 (RP11 BAC strains with -380E18, RP11-70N14, RP11-377D9) and c-kit (RP11-103A16, RP11-178P22) genes were streaked on agar plates containing antibiotics (chloramphenicol), and cultured overnight at 37°C (about 14 hours).

[0091] 2) Initial culture: Pick a monoclonal strain with good growth the next day, inoculate it in 2ml of LB liquid medium containing antibiotics (chloramphenicol), put it in a shaker at 37°C, set the shaker speed to about 200rpm, and culture 6-8 hours.

[0092] 3) Overnight culture: Transfer each 1ml of the bacterial solution obtained in step 2) to 200ml of LB liquid medium containing antibiotics (chloramphenicol), place i...

Embodiment 3

[0137] A probe kit (20 copies / box) for detecting the copy number change of the target gene in t(8;21) AML, including the following components:

[0138] 1) The ready-to-use hybridization solution obtained in step 3) in Example 2, in which the concentration of AML1 gene probe, ETO gene probe, WT1 gene probe, P27 gene probe and C-kit gene probe is 4ng / μl, a total of 200 μl.

[0139] 2), DAPI counterstaining solution 2ml.

[0140] 3), 1 box of 12*12mm cover glass.

[0141] 4), 1 box of 18*18mm cover glass.

[0142] 5), 1 bottle of liquid rubber.

[0143] 6), 1 box of glass slides.

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Abstract

The invention relates to a probe kit for detecting target gene copy-number alteration in t(8;21) AML. The probe kit includes a ready-to-use hybridization solution which comprises an AML1 gene probe, an ETO gene probe, a WT1 gene probe, a P27 gene probe and a C-kit gene probe, wherein the concentrations of all the gene probes are 4 ng / mu L. The probe kit provided by the invention has high combined probe signal intensity and strong stability and can realize qualitative and quantitative detection of changes of 5 target genes at a single cell level at the same time.

Description

technical field [0001] The invention relates to a detection kit, in particular to a probe kit for detecting the change of the copy number of the target gene in t(8;21) AML by using high-resolution quantitative multicolor fluorescence in situ hybridization, especially for detecting The evolutionary relationship between clones and the evolution of dominant clones and the growth and decline of subclones under the action of chemotherapy drugs. The combined probe signal intensity provided by the present invention is high, and stability is strong, and in DAPI (SP-100) of Chroma company, Spectrum Green TM (MF101),Cy3 TM v1(SP-102), Texas Red(SP-107) and Zeiss Cy5(50), PF-415(45) have no signal crossing under the six filter channels, and can be qualitatively and quantitatively detected at the single cell level at the same time5 Changes in a target gene. It can be used to detect changes in the copy number of target genes, tumor heterogeneity and subclonal evolution, and to detect th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6841C12Q2537/16C12Q2563/107
Inventor 王建祥王英婵胡林萍程涛
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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