Simultaneous Detection of FISH Signals and Bromo-Deoxyuridine Incorporation in Fixed Tissue Cultured Cells

FISH (Fluorescence in situ hybridization) is a powerful technique that detects and localises specific DNA sequences on metaphase chromosomes, interphase nuclei or chromatin fibres. When coupled to BrdU (5-Bromo 2-deoxy-uridine) labeling of newly replicated DNA, the replication properties of different DNA sequences can be analysed. However, the technique for the detection of BrdU incorporation is time consuming, and relies on acidic pH buffer treatments, that prevent use of pH sensitive fluorochromes such as FITC (Fluoro-isothiocianate) during FISH. In this work, we describe a simplified protocol that allows the simultaneous detection of FISH signals and BrdU incorporation. Since the technique does not involve paraformaldehyde for cell fixation, or formamide for denaturation of the target DNA and in post-hybridisation washes, it represents a safer alternative to classical FISH techniques

Simultaneous visualization of FISH signals and bromo-deoxyuridine incorporation by formamide-free DNA denaturation.

The replication timing of different DNA sequences in the mammalian cell nucleus is a tightly regulated system, which affects important cellular processes such as genes expression, chromatin epigenetic marking, and maintenance of chromosome structure. For this reason, it is important to study the replication properties of specific sequences, to determine for example, if the replication timing varies in different tissues, or in the presence of specific reagents, such as hormones, or other biologically active molecules. In this chapter, we present a technique, which allows identification of specific DNA sequences by fluorescence in situ hybridization (FISH) and simultaneously analyses the incorporation of a thymidine analogue, 5-bromo-2-deoxyuridine (BrdU), to mark DNA replication. First, tissue culture cells are synchronized at the beginning of the S-phase. BrdU is then added, either at specific time-points during S-phase or during the whole of the cell cycle. After harvesting the cells, the chromosomal DNA is hybridized to FISH probes that identify specific DNA sequences; this is performed without the teratogen formamide normally used in FISH. Finally, the cell preparations are analysed with an epifluorescence microscope to determine if the sequence of interest incorporates BrdU and in which point of the S-phase