I would like to hear about the experiences of FISH-mappers out there
doing DNA fibre-FISH.
Our experience is that it is extremely variable. I have had batches of
test slides which were a dream to analyse, and have yet to get any more
which approach that. Often, after analysing the whole slide, we have
signals in all possible permuations!
Often we find that less than 10% of slides prepared by alkaline lysis
(Fidlerova et al., 1994) are worth even using for hybridisation. It
seems that DNA release is so rapid that most of the DNA is lost off the
slide. Treating the slides with poly-L-lysine or APES doesn't seem to
help. Any suggestions, discussion would be greatly appreciated, but
particularly addressing the following questions:
1. What method works best, and CONSISTENTLY?
2. How long did it take to get the technique working in your lab?
3. Does it give sensible results in experimental situations? (ie. can
you interpret the data without prior knowledge of the result?)
4. What are the critical factors in getting the technique to work?
Thanks in advance,
Margaret
Dr. Margaret A. Leversha
The Sanger Centre
Cambridge
