Protocol

Chromosome Orientation Fluorescence In Situ Hybridization (CO-FISH)

¹ Drug Discovery Department, Moffitt Cancer Center, Tampa, FL 33612, USA
² Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523, USA

³Corresponding author (Eli.Williams{at}moffitt.org)

INTRODUCTION

Chromosome orientation fluorescence in situ hybridization (CO-FISH) differs from standard FISH in its ability to make hybridizations strand-specific. The procedure works by culturing cells for a single round of replication in the presence of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU), thus incorporating BrdU into the newly synthesized daughter strands. The daughter strands are then specifically removed through nuclease digestion, leaving single-stranded chromosomal DNA, which is an ideal target for hybridization of single-stranded probes. Overall, the effect is to produce single-sided signals. CO-FISH allows the investigator to determine the relative orientation of two or more DNA sequences along a chromosome. With a suitable reference probe (such as a telomere), CO-FISH can also determine the absolute 5'-to-3' direction of a DNA sequence relative to the short-arm-to-long-arm axis of the chromosome. This protocol describes the CO-FISH procedure, which uses a peptide nucleic acid (PNA) probe.