BRIEF REPORT |
Correspondence to:
Thomas Liehr, Institut für Humangenetik, Kollegiengasse 10, D-07743 Jena, Germany. E-mail:
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Summary |
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Fluorescence in situ hybridization (FISH) on human chromosomes in meta- and interphase is a well-established technique in clinical and tumor cytogenetics and for studies of evolution and interphase architecture. Many different protocols for labeling the DNA probes used for FISH have been published. Here we describe for the first time the successful use of Photoprobe biotin-labeled DNA probes in FISH experiments. Yeast artificial chromosome (YAC) and whole chromosome painting (wcp) probes were tested.
(J Histochem Cytochem 51:549551, 2003)
Key Words: FISH, DNA labeling, Photoprobe system
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Introduction |
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THE STANDARD APPROACh applied in molecular cytogenetics is fluorescence in situ hybridization (FISH). This technique is in use in clinical and tumor cytogenetics and in studies on chromosomal evolution and on the architecture of the interphase (for review see
The Photoprobe biotin labeling system (Linaris; WertheimBettingen, Germany) is based on an aryl azide derivative of biotin with a positively charged spacer arm between the biotin and the azide group. Using this system, biotin is directly conjugated to the nucleic acid (single- or double-stranded) in just 20 min using heat. Incorporation is random and is not base-specific.
This approach has been tested successfully on microdissection-derived and DOP-PCR-amplified wcp probes (
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Protocol |
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Two µg probe DNA [in our case probes were amplified by DOP-PCR (
In summary, we show for the first time the suitability of Photoprobe biotin-labeled probes for FISH approaches. Optimal FISH results could be obtained in metaphase (Fig 1A1C) and interphase chromosomes (Fig 1D). The Photoprobe biotin-labeling system is simple to perform. Labeling can be done in less than 1 hr and the resulting FISH signals are more intense than labeling done with standard protocols. The latter might be due to the fact that in nick-translation and DOP-PCR, only one of the four nucleotides (mostly dUTP) is conjugated with biotin. Using the Photoprobe biotin labeling system a random and not base-specific chemical conjugation of the whole DNA strand is done, which seems to be more effective.
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Acknowledgments |
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Supported by the DFG (PO284/6-1), the INTAS (2143), the BLE (99HS039), and the Wilhelm Sander-Stiftung (99.105.1). The continuous support of the Carl Zeiss GmbH (Jena, Germany) is gratefully acknowledged.
Dr M. Rocchi (Bari, Italy) is acknowledged for YAC probes of chromosome 2.
Received for publication August 29, 2002; accepted November 20, 2002.
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