22 DNA Breakage Detection-FISH (DBD-FISH)
287
- The surface area: number of pixels. As DNA breakage increases, DNA
loops of nucleoids progressively relax from supercoiling and the surface of the signal increases.
- The mean fluorescence intensity: average pixel grey count.
- Whole fluorescence intensity: surface x mean fluorescence intensity.
This is the sum of the grey intensities of all pixels of the signal. Thus, it
corresponds to the whole amount of hybridised probe.
Note: The same whole fluorescence intensity value may be obtained by
a low surface with a high grey mean level, and by a great surface with a
low grey mean level.
Comments
Initially, we recommend incubation times of 2.5 min in the unwinding
solution. In our conditions, shorter times do not allow the diffusion of
the alkali into the deeper cells within the gel, whereas longer times
may produce much melted ssDNA that is extended from breaks very distant from the probed locus.
The unwinding step before lysis may ((catch" induced breaks immediately, without increasing the background breakage level by manipulation
or during incubation. Nevertheless, lysis can be carried out before the unwinding, e.g., to obtain additional data on chromatin structure, as in
sperm cells (Fig. 3d,e).
Areas of hybridisation (e.g., of an alphoid DXZllocus probe) appear as
clearly separate, non-contiguous, spots after a high dose of X-rays (Fig.
3h). Neither double-strand break frequency nor DNA fragmentation after
dehydration and gel drying can explain this. Possibly, the neutralising step
produces partial and irregular renaturation of the ssDNA motif generated
from a DNA break by the alkali. The renaturation should take place within
the ssDNA motif and with the adjacent complementary strand in short
areas of homology, so the melted DNA curls up and becomes tangled
into a ball, trapping regions of ssDNA that hybridise with the probe, thereby giving the appearance of a spot (Fig. 3h).
Acknowledgements. This work was supported by the Consejo de Seguridad Nuclear, grants
from FIS and SAF (Spain) and European Community (FIGH-CT-1999-00009, TELORAD).
287
- The surface area: number of pixels. As DNA breakage increases, DNA
loops of nucleoids progressively relax from supercoiling and the surface of the signal increases.
- The mean fluorescence intensity: average pixel grey count.
- Whole fluorescence intensity: surface x mean fluorescence intensity.
This is the sum of the grey intensities of all pixels of the signal. Thus, it
corresponds to the whole amount of hybridised probe.
Note: The same whole fluorescence intensity value may be obtained by
a low surface with a high grey mean level, and by a great surface with a
low grey mean level.
Comments
Initially, we recommend incubation times of 2.5 min in the unwinding
solution. In our conditions, shorter times do not allow the diffusion of
the alkali into the deeper cells within the gel, whereas longer times
may produce much melted ssDNA that is extended from breaks very distant from the probed locus.
The unwinding step before lysis may ((catch" induced breaks immediately, without increasing the background breakage level by manipulation
or during incubation. Nevertheless, lysis can be carried out before the unwinding, e.g., to obtain additional data on chromatin structure, as in
sperm cells (Fig. 3d,e).
Areas of hybridisation (e.g., of an alphoid DXZllocus probe) appear as
clearly separate, non-contiguous, spots after a high dose of X-rays (Fig.
3h). Neither double-strand break frequency nor DNA fragmentation after
dehydration and gel drying can explain this. Possibly, the neutralising step
produces partial and irregular renaturation of the ssDNA motif generated
from a DNA break by the alkali. The renaturation should take place within
the ssDNA motif and with the adjacent complementary strand in short
areas of homology, so the melted DNA curls up and becomes tangled
into a ball, trapping regions of ssDNA that hybridise with the probe, thereby giving the appearance of a spot (Fig. 3h).
Acknowledgements. This work was supported by the Consejo de Seguridad Nuclear, grants
from FIS and SAF (Spain) and European Community (FIGH-CT-1999-00009, TELORAD).