Chapter 22
PROTOCOL
DNA Breakage Detection-FISH (DBD-FISH)
JOSE LUIS FERNANDEZ, VICENTE GOYANES, and JAIME GOSALVEZ
Introduction
DBD-FISH (DNA Breakage Detection- FISH) allows DNA breaks in single
cells to be detected and quantified, either in the whole genome or within
specific DNA sequence areas. This simple technique couples microgel embedding procedures (Fairbain et al. 1995), alkali unwinding assays (Rydberg 1975) and FISH.
Cells trapped within an agarose matrix over a slide are immersed in an
alkaline unwinding solution that produces single-stranded DNA (ssDNA)
motifs beginning from the ends of the molecule or internal strand breaks.
After neutralisation and protein removal in lysing solutions, the microgel
is dehydrated and cells incubated with DNA probes. The amount of hybridised probe in a target sequence is related to the amount of melted
ssDNA generated by the unwinding step, which is in turn proportional
to the degree of local DNA breakage.
Outline
The general steps for DBD-FISH are outlined in Fig. 1. This is a flexible
technique, allowing several variants depending on the experimental pur~ Jose Luis Fernandez, Centro Oncol6gico de Galicia, Laboratorio de Genetica
Molecular y Radiobiologia, Avda. de Montserrat sin, La Coruna, 15009, Spain
(phone +34-981-287499;fax +34-981-287122; e-mail genetica@cog.es, and Unidad de la
Mujer H. USP Santa Teresa, La Coruna, Spain)
Vicente Goyanes, Hospital Materno Infantil Teresa Herrera, Departamento de
Genetica, La Corufta, 15009, Spain, and Unidad de la Mujer H. USP Santa Teresa,
La Coruna, Spain
Jaime Gosalvez, Universidad Aut6noma de Madrid, Unidad de Genetica, Facultad de
Biologia, Madrid, 28049, Spain
PROTOCOL
DNA Breakage Detection-FISH (DBD-FISH)
JOSE LUIS FERNANDEZ, VICENTE GOYANES, and JAIME GOSALVEZ
Introduction
DBD-FISH (DNA Breakage Detection- FISH) allows DNA breaks in single
cells to be detected and quantified, either in the whole genome or within
specific DNA sequence areas. This simple technique couples microgel embedding procedures (Fairbain et al. 1995), alkali unwinding assays (Rydberg 1975) and FISH.
Cells trapped within an agarose matrix over a slide are immersed in an
alkaline unwinding solution that produces single-stranded DNA (ssDNA)
motifs beginning from the ends of the molecule or internal strand breaks.
After neutralisation and protein removal in lysing solutions, the microgel
is dehydrated and cells incubated with DNA probes. The amount of hybridised probe in a target sequence is related to the amount of melted
ssDNA generated by the unwinding step, which is in turn proportional
to the degree of local DNA breakage.
Outline
The general steps for DBD-FISH are outlined in Fig. 1. This is a flexible
technique, allowing several variants depending on the experimental pur~ Jose Luis Fernandez, Centro Oncol6gico de Galicia, Laboratorio de Genetica
Molecular y Radiobiologia, Avda. de Montserrat sin, La Coruna, 15009, Spain
(phone +34-981-287499;fax +34-981-287122; e-mail genetica@cog.es, and Unidad de la
Mujer H. USP Santa Teresa, La Coruna, Spain)
Vicente Goyanes, Hospital Materno Infantil Teresa Herrera, Departamento de
Genetica, La Corufta, 15009, Spain, and Unidad de la Mujer H. USP Santa Teresa,
La Coruna, Spain
Jaime Gosalvez, Universidad Aut6noma de Madrid, Unidad de Genetica, Facultad de
Biologia, Madrid, 28049, Spain