Chapter 21
PROTOCOL
The Combined Use of Nuclease in Situ Digestion
and FISH (NU-FISH)
JAIME GOSALVEZ, JOAQUINA DE LA TORRE,
CARMEN LOPEZ-FERNANDEZ, and JosE LUIS FERNANDEZ
Introduction
Metaphase chromosomes or even interphase nuclei are highly organised
structures made up of proteins and DNA and have a high degree of complex packaging. However, this does not preclude the "in situ" cleavage of
DNA on conventionally fIxed cytological preparations with active nucleases, i.e. fIXed chromatin is susceptible to "in situ" digestion. DNA extraction can be monitored by staining the cytological preparations with
DNA-specifIc dyes. This approach may furnish information on the presence/absence of enzyme receptive sites along the chromosomes. The removal of specifIc DNA fragments results in the production of a chromosome banding pattern, which can be used to analyse the differences between and resemblance of distinct chromosomal regions and/or of genomes of related species.
After almost 15 years of experience using endo- and exo-nucleases to
induce the selective removal of DNA from fIxed and unfixed chromatin,
we know that DNA extractability depends on a series of factors (L6pezFernandez et at. 1991; de la Torre and Sumner 1994). All of them must be
carefully evaluated before comparing the results obtained from slightly
different experimental conditions. However, the interpretation of the results obtained after nuclease "in situ" digestion and post-staining with
DNA-specifIc dyes is far from straightforward. This situation substantially
~ Jaime GosaIvez, Universidad Autonoma de Madrid, Departamento de Biologia,
Unidad de Genetica, Madrid, 28049, Spain (phone +34-91-397-8210;
fax +34-91-397-8344; e-mail jaime.gosalvez@uam.es)
Joaquina de la Torre, Universidad Autonoma de Madrid, Departamento de Biologia,
Unidad de Genetica, Madrid, 28049, Spain
Carmen Lopez-Fernandez, Universidad Autonoma de Madrid, Departamento
de Biologia, Unidad de Genetica, Madrid, 28049, Spain
Jose Luis Fernandez, Centro Oncologico de Galicia, Unidad de Genetica, Avenida
PROTOCOL
The Combined Use of Nuclease in Situ Digestion
and FISH (NU-FISH)
JAIME GOSALVEZ, JOAQUINA DE LA TORRE,
CARMEN LOPEZ-FERNANDEZ, and JosE LUIS FERNANDEZ
Introduction
Metaphase chromosomes or even interphase nuclei are highly organised
structures made up of proteins and DNA and have a high degree of complex packaging. However, this does not preclude the "in situ" cleavage of
DNA on conventionally fIxed cytological preparations with active nucleases, i.e. fIXed chromatin is susceptible to "in situ" digestion. DNA extraction can be monitored by staining the cytological preparations with
DNA-specifIc dyes. This approach may furnish information on the presence/absence of enzyme receptive sites along the chromosomes. The removal of specifIc DNA fragments results in the production of a chromosome banding pattern, which can be used to analyse the differences between and resemblance of distinct chromosomal regions and/or of genomes of related species.
After almost 15 years of experience using endo- and exo-nucleases to
induce the selective removal of DNA from fIxed and unfixed chromatin,
we know that DNA extractability depends on a series of factors (L6pezFernandez et at. 1991; de la Torre and Sumner 1994). All of them must be
carefully evaluated before comparing the results obtained from slightly
different experimental conditions. However, the interpretation of the results obtained after nuclease "in situ" digestion and post-staining with
DNA-specifIc dyes is far from straightforward. This situation substantially
~ Jaime GosaIvez, Universidad Autonoma de Madrid, Departamento de Biologia,
Unidad de Genetica, Madrid, 28049, Spain (phone +34-91-397-8210;
fax +34-91-397-8344; e-mail jaime.gosalvez@uam.es)
Joaquina de la Torre, Universidad Autonoma de Madrid, Departamento de Biologia,
Unidad de Genetica, Madrid, 28049, Spain
Carmen Lopez-Fernandez, Universidad Autonoma de Madrid, Departamento
de Biologia, Unidad de Genetica, Madrid, 28049, Spain
Jose Luis Fernandez, Centro Oncologico de Galicia, Unidad de Genetica, Avenida