Abstract
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.
Highlights
The replication of DNA in eukaryotic cells is tightly regulated and time controlled
We describe an alternative technique which allows the simultaneous detection of BrdU incorporation and FISH signals
We developed an efficient method to analyse the replication timing of a human artificial chromosome (HAC) containing chromosome 17 alphoid DNA [10,11] in human (AG61) and murine (LAMF4-5) cells, using bromo-deoxyuridine incorporation and FISH signals in fixed tissue cultured cells
Summary
The replication of DNA in eukaryotic cells is tightly regulated and time controlled. Euchromatic regions, containing actively expressed genes, are generally replicated in the early stages of the S phase, while non active genes preferentially cluster in areas that are replicated in later stages [1]. The active copy of the X chromosome is replicated alongside the other autosomes, depending on its gene content, but the inactive copy is the last chromosome to be replicated in the S phase [2]. It has been observed that the difference in replication timing often corresponds to a different spatial localisation of DNA sequence in the nuclear architecture [1]. Incorrect replication timing of characteristic sequences has been associated with some human diseases such as DiGeorge, Velocardiofacial and Roberts syndromes [3,4]
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