Chromosomics: Определение численного и структурные изменения во всех 24 хромосом человека одновременно, используя Роман OctoChrome Анализ FISH
Summary
Роман флуоресценции<em> На месте</em> Гибридизации (FISH), который одновременно рассматривается как численные и структурные изменения хромосом, в частности, специфической хромосомной транслокации связанных с лейкемией и лимфомой, из всех 24 хромосом человека на одном устройстве в одной гибридизации, описан.
Abstract
Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis.
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously.
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.
Protocol
Discussion
Этот роман OctoChrome-FISH анализ позволяет одновременно изучить все 24 хромосом человека в одной гибридизации на одном слайде. Расположение хромосом комбинации на 8 квадратов был разработан, чтобы облегчить идентификацию неслучайных хромосомных перестроек в наиболее распространенных лейк?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
Авторы хотели бы поблагодарить д-ра М. Cliona Макхейл для критического чтения и редактирования рукописей. Эта работа финансировалась Национальным институтом экологических наук, Национальный институт здравоохранения грант R01ES017452 в L Чжан.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| OctoChrome Kit | Cytocell Ltd, Cambridge, UK | PMP 803 | |
| phytohaemagglutinin (PHA) | Invitrogen Corporation | 10576-015 | |
| Colcemid | Invitrogen Corporation | 15212-012 | |
| Carnoy’s fixative | Methanol : glacial acetic acid = 3: 1 | ||
| Fluorescence microscope | Equipped with filters to view DAPI, Texas Red, FITC, and Coumarin spectra individually and a DAPI/FITC/Texas Red triple filter to view different colors simultaneously | ||
| Metafer software | MetaSystems, Altlussheim, Germany | Facilitate to locate all metaphases and to re-evaluate the abnormalities |
Referencias
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