21 The Combined Use of Nuclease in Situ Digestion and FISH (NU-FISH)
275
Chromosomes obtained from different sources (insects, fish, amphibia, Notes
anura, birds, mammals and plants) have been used by different authors.
Preparations are obtained following standard procedures of fixation and
current techniques of spreading and squashing. Unfixed chromatin is also
susceptible to RE-«in situ" digestion (Burkholder 1989, GosaIvez et al.
1993). Standard prepared chromosomes or chromatin fibre can be used.
Any RE has the potential to produce selective DNA cleavage on fixed
chromatin and, in turn, to provoke DNA loss. However, since DNA removal is more effective when short DNA fragments are produced, the
use of four, five or six DNA base pair cutters is preferable. In fact, after
cleaving with REs with more than 6 bp targets, long DNA fragments are
produced that remain tangled in the chromatin mass.
Modifying enzymes
Modifying enzymes (ME) alter the DNA or RNA according to a specific
polynucleotide structure. So far, only Exonuclease III and Bal31 have been
used in combination with REs, for «in situ" hybridisation purposes.
Exonuclease III (Exo III) produces a stepwise removal of nucleotides from Exonuclease III
the 3'-ends of double-stranded DNA, thus giving rise to single-stranded
DNA that can be used as a target for DNA probing.
1. Chromosomal DNA is nicked with REs according to the subprotocol described for RE-digestion but using shorter incubation times
(15-30 min).
2. After RE digestion, the slides are washed in 2x SSC (pH 7), dehydrated
in an ethanol series (70%, 85%, 100%) and air dried.
3. The slides are incubated with 150U of ExoIII in 50 III of buffer for
10 min at 37°C.
4. After ExoIII digestion, the slides are washed in 2xSSC (pH 7), dehydrated in an ethanol series (70%,85%, 100%) and air dried.
The action of ExoIII produces enzymatic denaturation of DNA; thus, sin- Notes
gle-stranded DNA (ssDNA) areas will already be available for «in situ"
hybridisation of selected probes, Le., no further heat denaturation of
the chromosomes will be required.
275
Chromosomes obtained from different sources (insects, fish, amphibia, Notes
anura, birds, mammals and plants) have been used by different authors.
Preparations are obtained following standard procedures of fixation and
current techniques of spreading and squashing. Unfixed chromatin is also
susceptible to RE-«in situ" digestion (Burkholder 1989, GosaIvez et al.
1993). Standard prepared chromosomes or chromatin fibre can be used.
Any RE has the potential to produce selective DNA cleavage on fixed
chromatin and, in turn, to provoke DNA loss. However, since DNA removal is more effective when short DNA fragments are produced, the
use of four, five or six DNA base pair cutters is preferable. In fact, after
cleaving with REs with more than 6 bp targets, long DNA fragments are
produced that remain tangled in the chromatin mass.
Modifying enzymes
Modifying enzymes (ME) alter the DNA or RNA according to a specific
polynucleotide structure. So far, only Exonuclease III and Bal31 have been
used in combination with REs, for «in situ" hybridisation purposes.
Exonuclease III (Exo III) produces a stepwise removal of nucleotides from Exonuclease III
the 3'-ends of double-stranded DNA, thus giving rise to single-stranded
DNA that can be used as a target for DNA probing.
1. Chromosomal DNA is nicked with REs according to the subprotocol described for RE-digestion but using shorter incubation times
(15-30 min).
2. After RE digestion, the slides are washed in 2x SSC (pH 7), dehydrated
in an ethanol series (70%, 85%, 100%) and air dried.
3. The slides are incubated with 150U of ExoIII in 50 III of buffer for
10 min at 37°C.
4. After ExoIII digestion, the slides are washed in 2xSSC (pH 7), dehydrated in an ethanol series (70%,85%, 100%) and air dried.
The action of ExoIII produces enzymatic denaturation of DNA; thus, sin- Notes
gle-stranded DNA (ssDNA) areas will already be available for «in situ"
hybridisation of selected probes, Le., no further heat denaturation of
the chromosomes will be required.