31 Multicolor FISH Techniques for the Detection of Inter- and Intrachromosomal Rearrangements
423
Fig. 4. The der(21) in TPC-l carries material from the
long arm of chromosome 10 from ret (probe S8Cll,
purple) to qter. The characteristic ret-H4/DIOSl70
inversion is indicated by the arrows (C)
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- If the hybridization signal is weak in CR FISH, put slide under light
protected conditions in the refrigerator for one day after detection
and check it again under the microscope.
- Random priming reactions reveal better incorporation for CR FISH
applications of labeled nucleotides than nick translation; if you decide
to nick translate your probes, check the fragment sizes after nick translation by agarose gel electrophoresis. If an optimum range of fragment
distribution between 300-1000 bp is not present, digest additionally
with DNase I (time and concentration of incubation must be figured
out experimentally). An improved incorporation of labeled nucleotides can also be obtained by using two different labeled nucleotides
(e.g., biotinylated dUTP and dCTP) in the reaction.
Acknowledgements. This work was supported in part by the German Bundesamt fUr Wehrtechnik und Beschaffung, contract number E/B41GIV0132/TS922. Further support was provided by the US Department of Energy under contract DE-AC-03-76SF00098 and grants
from the National Institute of Health (lR2l CA882S8, IROI CA80792).