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Titolo:
New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling
Autore:
Tanke, HJ; Wiegant, J; van Gijlswijk, RPM; Bezrookove, V; Pattenier, H; Heetebrij, RJ; Talman, EG; Raap, AK; Vrolijk, J;
Indirizzi:
Leiden Univ, Med Ctr, Dept Mol Cell Biol, Lab Cytochem & Cytometry, NL-2333 Leiden Univ Leiden Netherlands NL-2333 AL Cytochem & Cytometry, NL-2333 LeidensUniv, Leiden Inst Chem, Gorlaeus Labs, NL-2300 RA Leiden, Netherland Leiden Univ Leiden Netherlands NL-2300 RA NL-2300 RA Leiden, Netherland
Titolo Testata:
EUROPEAN JOURNAL OF HUMAN GENETICS
fascicolo: 1, volume: 7, anno: 1999,
pagine: 2 - 11
SICI:
1018-4813(199901)7:1<2:NSFMFI>2.0.ZU;2-W
Fonte:
ISI
Lingua:
ENG
Soggetto:
DIGITAL IMAGING MICROSCOPY; INSITU HYBRIDIZATION; HUMAN-CHROMOSOMES; DNA PROBES; KARYOTYPE; PCR;
Keywords:
cytogenetics; chromosomal aberrations; multicolour FISH; combinatorial labelling; chemical labelling; ratio labelling; digital fluorescence microscopy;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
17
Recensione:
Indirizzi per estratti:
Indirizzo: Tanke, HJ LeidenarsewegMed Ctr, Dept Mol Cell Biol, Lab Cytochem & Cytometry, Wassena Leiden Univ Wassenaarseweg 72 Leiden Netherlands NL-2333 AL ena
Citazione:
H.J. Tanke et al., "New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling", EUR J HUM G, 7(1), 1999, pp. 2-11

Abstract

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 aset 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.

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Documento generato il 28/11/20 alle ore 15:49:46