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| MULTIPLE AND SENSITIVE FLUORESCENCE INSITU HYBRIDIZATION WITH RHODAMINE-LABELED, FLUORESCEIN-LABELED, AND COUMARIN-LABELED DNAS |
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| Author(s): WIEGANT J, WIESMEIJER CC, HOOVERS JMN, SCHUURING E, DAZZO A, VROLIJK J, TANKE HJ, RAAP AK |
| Source: CYTOGENETICS AND CELL GENETICS Volume: 63 Issue: 1 Pages: 73-& Published: 1993 |
| Times Cited: 63 References: 26 |
| Abstract: We have tested the use of several newly developed red, green, and blue fluorescent dUTPs in direct, multiple, and sensitive fluorescence in situ hybridization procedures. Among the ones tested, the tetramethylrhodamine-dUTP proved to give the best sensitivity; using conventional epifluorescence microscopy, cosmids could be visualized with a hybridization efficiency of 90%. Fluorescein-dUTP permitted visual cosmid detection with 50% efficiency, and, to the human eye, the green signals appeared less bright. With a blue fluorescent coumarin-labeled dUTP, only highly repetitive target sequences could be visualized directly. Imaging with a cooled CCD (charged coupled device) camera for prolonged integration times permitted localization of small targets using probes labeled with red and green fluorochromes. Visualization of a 1.8-kb single copy sequence with a rhodamine-labeled cDNA proved feasible. The strategy of identifying hybridization sites with multiple colors was successfully applied for multiple hybridizations to repetitive and unique targets, using the red and green labels directly and the biotin label indirectly with one layer of avidin-coumarin. |
| Document Type: Article |
| Language: English |
| Reprint Address: WIEGANT, J (reprint author), LEIDEN UNIV, DEPT CYTOCHEM & CYTOMETRY, WASSENAARSEWEG 72, 2333 AL LEIDEN, NETHERLANDS |
Addresses:
1. LEIDEN UNIV, DEPT PATHOL, 2333 AL LEIDEN, NETHERLANDS
2. DEPT CELL BIOL & GENET, ROTTERDAM, NETHERLANDS
3. UNIV AMSTERDAM, DEPT HUMAN GENET, AMSTERDAM, NETHERLANDS |
| Publisher: KARGER, ALLSCHWILERSTRASSE 10, CH-4009 BASEL, SWITZERLAND |
| Subject Category: Cell Biology; Genetics & Heredity |
| IDS Number: KW929 |
| ISSN: 0301-0171 |
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