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Method and system for analyzing chromosomal translocation

A technique for chromosomal translocation and sample analysis, applied in the field of systems for analyzing chromosomal translocations

Active Publication Date: 2016-07-06
VENTANA MEDICAL SYST INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, none of these systems have been applied to address the problems associated with false positive results in split probe systems

Method used

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  • Method and system for analyzing chromosomal translocation
  • Method and system for analyzing chromosomal translocation
  • Method and system for analyzing chromosomal translocation

Examples

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Embodiment 1

[0139] Materials and methods

[0140] ALK Triple Probe Design

[0141] The breakaway in situ hybridization (ba-ISH) assay was designed to assess rearrangements at the ALK gene locus (ALK-EML4 fusion). Three probes were generated to hybridize to the adjacent centromere region (770kb) and telomeric region (683kb) of the ALK gene as well as the ALK gene region (728Kb) ( figure 1 ). The ALK gene probe was labeled with the NP hapten, the 5' ALK probe was labeled with the DIG hapten, and the 3' ALK probe was labeled with the DNP hapten.

[0142] Automated Brightfield Separation In Situ Hybridization Protocol

[0143] All optimization and performance evaluations of the brightfield in situ hybridization ALK gene ba-ISH assay were performed using XT automated slide processing system (Ventana Medical Systems, Inc., Tucson, AZ). Create ba-ISH instrument software so that all steps from baking to counterstaining can be performed without interruption. Slides were baked on the instrum...

Embodiment 2

[0145]The following examples describe methods for the analysis of ALK rearrangements using a three-color separation probe system. Slides were baked on the instrument at 65°C for 20 minutes to melt the paraffin, followed by an EZ preparation (Ventana Medical Systems, Inc.) deparaffinization step initiated by liquid coverslips (Ventana Medical Systems, Inc.). DNA targets were recovered by a combination of heat treatment with citrate buffer-based target recovery solution CC2 (Ventana Medical Systems, Inc.) and tissue digestion with ISH protease 3 (Ventana Medical Systems, Inc.). Due to the different tissue fixation and processing conditions of clinical samples, determine the appropriate protease digestion time for each tissue sample. A mixture of 3 ALK probes (DNP-labeled 5' ALK probe, fluorescein-labeled internal ALK probe, and DIG-labeled 3' ALK probe, 12 μg / ml each) was mixed with Ventana hybridization buffer prepared with fish DNA. Probe and target DNA were co-denatured at ...

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Abstract

The present disclosure relates to systems and methods for analyzing chromosomal translocations, and in particular to analysis of chromosomal translocations by in situ hybridization. The method employs a 5' probe, a 3' probe, and a probe that binds near the breakpoint, and optionally, overlaps the breakpoint. The probes are labeled differently. In particular, use in detecting translocations involving the ALK gene is described.

Description

field of invention [0001] The present disclosure relates to systems and methods for analyzing chromosomal translocations, and in particular to analysis of chromosomal translocations by in situ hybridization. Background of the invention [0002] Diagnosis, prognosis, and determination of treatment of disease based on the interpretation of tissue or cell samples taken from diseased organisms has expanded dramatically over the past few years. In addition to traditional histological staining techniques and immunohistochemical assays, in situ techniques such as in situ hybridization and in situ polymerase chain reaction are now used to aid in the diagnosis of human disease states. As such, a variety of techniques exist that can assess not only cell morphology but also the presence of specific macromolecules within cells and tissues. [0003] Molecular cytogenetic techniques such as chromogenic in situ hybridization (CISH) combine visual assessment of chromosomes (karyotyping) wi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6841C12Q2565/102C12Q2539/115C12Q1/6827C12Q2525/185C12Q2543/101
Inventor M.法雷尔T.M.格罗甘H.尼塔张文军
Owner VENTANA MEDICAL SYST INC