New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling
New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling
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多色荧光原位杂交新策略：COBRA：CObined Binary RAtio 标记
DOI：
10.1038/sj.ejhg.5200265
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发表时间：
1999
影响因子：
5.2
通讯作者：
J. Vrolijk
中科院分区：
文献类型：
--
作者：
H. J. Tanke;J. Wiegant;R. V. Gijlswijk;V. Bezrookove;H. Pattenier;R. J. Heetebrij;E. G. Talman;A. Raap;J. Vrolijk
多色原位杂交(MFISH)越来越多地应用于染色体核型分析和染色体异常的检测。到目前为止，已经用所谓的组合方法使用荧光标记的染色体绘制探针描述了27种颜色分析。本文提出了一种新的策略，以有效地利用目前可用的光谱分离的荧光团的数量，以增加MFISH的多样性。介绍了基于组合标记法和比率标记法同时使用的组合二进制比率标记法的原理。人类染色体24种颜色的绘制仅使用四个荧光团即可完成。三个荧光团用于一组12个染色体涂抹探针的比例标记。第二组12个探针被相同地标记，但也被给予二元标记(第四荧光团)。COBRA方法在正常人染色体和淋巴瘤(JVM)细胞系上进行了演示，以荧光素、利沙明和Cy5酶标记的探针为主要荧光团，以蓝色染料二乙氨基香豆素(DEAC)为组合第四标记，以证明地高辛参入。此外，使用化学标记对这一原理进行了测试。因此，第一组12个涂抹探针被ULS(Universal Linkage System)标记，使用DEAC、Cy3和Cy5作为主标记，第二组类似地高辛标记，但也包含地高辛-ULS标记，其间接地被荧光素染色。随后，给出了数学分析，并指出了使用现有技术实现48、96或更高的MFISH多重数的方法。
Multicolour in situ hybridisation (MFISH) is increasingly applied to karyotyping and detection of chromosomal abnormalities. So far 27 colour analyses have been described using fluorescently labelled chromosome painting probes in a so-called combinatorial approach. In this paper a new strategy is presented to use efficiently the currently available number of spectrally separated fluorophores in order to increase the multiplicity of MFISH. We introduce the principle of COBRA (COmbined Binary RAtio labelling), which is based on the simultaneous use of combinatorial labelling and ratio labelling. Human chromosome painting in 24 colours is accomplished using four fluorophores only. Three fluorophores are used pair wise for ratio labelling of a set of 12 chromosome painting probes. The second set of 12 probes is labelled identically but is also given a binary label (fourth fluorophore). The COBRA method is demonstrated on normal human chromosomes and on a lymphoma (JVM) cell line, using probes enzymatically labelled with fluorescein, lissamine and cy5 as primary fluorophores, and diethylaminocoumarin (DEAC), a blue dye, as combinatorial fourth label to demonstrate incorporated digoxigenin. In addition, the principle was tested using chemical labelling. The first set of 12 painting probes was therefore labelled by ULS (Universal Linkage System), using DEAC, cy3 and cy5 as primary labels, and the second set was labelled similarly, but also contained a digoxigenin-ULS label, which was indirectly stained with fluorescein. Subsequently, a mathematical analysis is presented and methods are indicated for achieving an MFISH multiplicity of 48, 96 or even higher using existing technology.
DOI：
10.1073/pnas.89.4.1388
发表时间：
1992-02-15
影响因子：
11.1
作者：
RIED, T;BALDINI, A;WARD, DC
通讯作者：
WARD, DC