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Compositions and methods for hybridization

A technology of composition and hybridization probe, which is applied in the field of nucleic acid and can solve the problem of expensive hybridization probes

Active Publication Date: 2021-05-11
42 LIFE SCI GMBH & CO KG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since hybridization probes are very expensive, there have hitherto been no methods known from the prior art or no hybridization compositions known from the prior art for automated in situ hybridization which is also economically possible.

Method used

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  • Compositions and methods for hybridization
  • Compositions and methods for hybridization
  • Compositions and methods for hybridization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment A1

[0172] Example A.1 ISH Procedure I (flexible FISH with hybridization duration of two hours or 16 hours)

[0173] Reagents for performing "flexible" rapid FISH were from the ZytoLight Flexible FISH-Tissue Performance Kit (Z-2182-20, ZytoVision GmbH, Bremerhaven, Germany). The kit contains the necessary reagents for performing FISH on formalin-fixed and paraffin-embedded tissue sections (heat-pretreated citric acid solution, pepsin solution, 5x concentrated flexible FISH wash buffer, and DAPI / DuraTect TM solution).

[0174] FISH was performed on 3 to 5 μm thick sections of formalin-fixed paraffin-embedded (FFPE) tissue from breast cancer, lung tissue, lymph node tissue, kidney tissue, prostate tissue, and placenta tissue. Tissue sections were mounted on coated glass slides and baked at 58°C overnight.

[0175] To remove paraffin, preparations were incubated twice in 100% xylene at room temperature (RT) for five minutes each. Thereafter, a series of decreasing ethanol for two ...

Embodiment A2

[0179] Example A.2 ISH Procedure II (Automated FISH on an automated system Celerus Wave RPD system)

[0180] Reagents for performing automated FISH (on an automated system Celerus Wave RPD system from Celerus Diagnostics, California, USA) were contained in the LRM Bin (Celerus Diagnostics, California, USA). LRM (Linear Reagent Magazine) contains all necessary reagents for heat pretreatment, proteolysis and wash buffers for required washing steps. DAPI / DuraTect TM (ZytoVision GmbH, Bremerhaven, Germany) was used to overlay the preparation and stain the nuclei. Automated system probes are positioned in the PAC Bin inserted in the LRMBin.

[0181] Automated FISH is performed on 3 to 5 μm thick formalin-fixed and paraffin-embedded (FFPE) human tissue sections obtained from mammalian cancer, lung, lymph node, kidney, prostate, and / or placenta, which are mounted on Coated glass slides were baked overnight at 58 °C. To remove paraffin, according to Celerus The RPD system's pred...

Embodiment A3I

[0185] Example A.3 ISH Procedure III (Automated FISH on Automated Systems Pathcom Stainer)

[0186] Reagents used to perform automated FISH (on automated systems Pathcom stainers from PathCom Systems Cooperation, California, USA) were from the ISH detection kit (PathCom Systems Corporation, Sierra Ct, Dublin, USA). Kit contains all necessary reactions for performing automated FISH (deparaffin solution, repair solution, pepsin, diH 2 O). Additionally, PathCom System Wash Buffers for IHC and ISH are required. DAPI / DuraTect TM Solution (ZytoVision GmbH, Bremerhaven, Germany) was used to cover the preparation and stain the nuclei.

[0187] Automated FISH is performed on 3 to 5 µm thick formalin-fixed and paraffin-embedded (FFPE) human tissue sections, such as those from mammalian cancers, lungs, lymph nodes, kidneys, prostate, and / or placenta, mounted on Coated glass slides were baked overnight at 58 °C.

[0188] To remove paraffin, the formulation was initially treated with ...

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Abstract

The present invention relates to a composition for hybridization comprising: (a) at least one hybridization probe ("component (a)"); (b) at least one polar probe having a circular molecular structure protic or polar aprotic solvent ("component (b)"); and (c) at least one carboxylic acid amide and / or its salt ("component (c)"), with respect to the composition greater than 10vol% amount. Furthermore, the present invention relates to the use of such a composition, as well as a method for detecting nucleic acid and / or chromosomal abnormalities in a biological sample via hybridization using said composition.

Description

Background technique [0001] The present invention is directed to the technical field of methods for the detection of nucleic acids, in particular DNA and / or RNA. [0002] In particular, the invention relates to a composition for hybridization and in particular for the identification and / or detection of nucleic acids, and its use according to the invention. Furthermore, the present invention relates to a method for detecting nucleic acid or chromosomal abnormalities. Finally, the invention relates to a kit for detecting nucleic acid or chromosomal abnormalities. [0003] Many neoplastic diseases are based on structural and numerical chromosomal mutations, such as translocations, inversions, segmental duplications, deletions, insertions, duplications, aneuploidies, and amplifications. These changes are typically detected via in situ hybridization (ISH) as predictive, prognostic or differential diagnostic markers. [0004] In situ hybridization is based on the hybridization or...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6832C12Q1/6841
CPCC12Q1/6841C12Q2527/125C12Q2527/137C12Q1/6832
Inventor 皮尔·马格拉夫-罗加拉斯文·豪克
Owner 42 LIFE SCI GMBH & CO KG
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