Chromosome Replicating Timing Combined with Fluorescent <em>In situ</em> Hybridization

Leslie Smith; Mathew Thayer

doi:10.3791/4400

Chromosome Replizieren Timing mit Fluorescent Kombiniert In situ Hybridisierung

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Biology

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Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Chromosome Replizieren Timing mit Fluorescent Kombiniert In situ Hybridisierung

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17:14 min

December 10, 2012

DOI: 10.3791/4400-v

Leslie Smith¹, Mathew Thayer¹

¹Department of Biochemistry and Molecular Biology, Knight Cancer Institute,Oregon Health & Science University

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Overview

This article describes a quantitative method for analyzing chromosome replication timing using BrdU incorporation and fluorescent in situ hybridization (FISH). The technique enables direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Key Study Components

Area of Science

Cell Biology
Genetics
Chromosome Dynamics

Background

Understanding chromosome replication timing is crucial for insights into genomic stability.
BrdU incorporation allows for the labeling of newly synthesized DNA.
FISH provides a method to visualize specific DNA sequences within chromosomes.
Combining these techniques enhances the analysis of chromosomal behavior during replication.

Purpose of Study

To develop a method for quantitatively assessing replication timing of mammalian chromosomes.
To compare the replication timing of rearranged versus un-rearranged chromosomes.
To improve understanding of chromosomal replication dynamics in live cells.

Methods Used

Incorporation of BrdU into live cells.
Harvesting of metaphase chromosomes for analysis.
Application of DNA FISH combined with sequential BrdU antibody staining.
Use of photomicroscopy and software for image analysis of FISH signals and BrdU incorporation.

Main Results

Successful identification of individual replicating chromosomes.
Quantitative data on replication timing differences between chromosome types.
Visual representation of chromosomal behavior during replication.
Insights into the implications of chromosomal rearrangements on replication timing.

Conclusions

The method provides a robust approach for studying chromosome replication timing.
Direct comparisons can enhance understanding of genomic stability.
This technique may have broader applications in genetic research.

Frequently Asked Questions

What is BrdU incorporation?

BrdU incorporation is a method used to label newly synthesized DNA, allowing researchers to track DNA replication.

How does FISH work?

Fluorescent in situ hybridization (FISH) uses fluorescent probes that bind to specific DNA sequences, enabling visualization of chromosomes.

What are the advantages of this method?

This method allows for direct comparisons of chromosome replication timing within the same cell, providing more accurate insights.

Can this technique be applied to other organisms?

While this study focuses on mammalian chromosomes, the principles may be adapted for use in other species.

What implications does this research have?

Understanding replication timing can shed light on genomic stability and the effects of chromosomal rearrangements.

Is this method suitable for clinical applications?

The technique may have potential clinical applications in cancer research and genetic disorders.

Ein quantitatives Verfahren zur Analyse von Chromosomenreplikation Timing beschrieben. Das Verfahren nutzt BrdU Inkorporation in Kombination mit fluoreszierenden

Dieser experimentelle Ansatz kombiniert den BRDU-Einbau mit der Fluoreszenz-in-C-Zwei-Hybridisierung. Quantitative Analyse des Replikationszeitpunkts von Säugetierchromosomen. Zuerst wird BRDU in lebende Zellen eingebaut und die Metaphase-Chromosomen geerntet.

Führen Sie dann DNA-Fische in Kombination mit sequentieller BRDU-Antikörperfärbung durch, um die einzelnen replizierenden Chromosomen zu identifizieren. Verwenden Sie als Nächstes die Fotomikroskopie, um Bilder von Fischsignalen und der BRDU-Integration in einzelne mitotische Figuren aufzunehmen. Berechnen Sie mit der Cyto-Vision-Software die Fläche und Intensität der Pixel, die durch die BRDU- oder DPI-Signale auf jedem isolierten Chromosom dargestellt werden.

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