Nonradioactive in Situ Hybridization with Digoxigenin Labeled DNA Probes
作者:
ArnoldN.,
SeiblR.,
KesslerC.,
WienbergJ.,
期刊:
Biotechnic&Histochemistry
(Taylor Available online 1992)
卷期:
Volume 67,
issue 2
页码: 59-67
ISSN:1052-0295
年代: 1992
DOI:10.3109/10520299209110009
出版商: Taylor&Francis
关键词: hybridocytochemistry;in situ hybridization;digoxigenin;chromosomes
数据来源: Taylor
摘要:
Nonradioactive in situ hybridization techniques are becoming increasingly important tools for rapid analysis of the topological organization of DNA and RNA sequences within cells. Prerequisite for further advances with these techniques are multiple labeling and detection systems for different probes. Here we summarize our results with a recently developed labeling and detection system. The DNA probe for in situ hybridization is modified with digoxigenin-labeled deoxyuridine-triphosphate. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Labeled DNA probes were hybridized in situ to chromosome preparations. The hybridization signal was detected using digoxigenin-specific antibodies covalently coupled to enzyme markers (alkaline phosphatase or peroxidase) or to fluorescent dyes. Color reactions catalyzed by the enzymes resulted in precipitates located on the chromosomes at the site of probe hybridization. This was verified by hybridizing DNA probes of known chromosomal origin. The signals were analyzed by bright field, reflection contrast and fluorescence microscopy. The results indicate that the new technique gives strong signals and can also be used in combination with other systems (e.g., biotin) to detect differently labeled DNA probes on the same metaphase plate.
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