FISH Background

RPSCOTT RSCOTT at IRRI.CGNET.COM
Tue Jan 26 06:45:10 EST 1993


Mike Morris at "mike at medsun.unige.ch" writes: 
>   We are doing fluorescent in situ hybridization on human chromosomes:
>single copy, painting, interphase, the lot.
>   Although we have no trouble (at last!) getting good signals and good
>specificity, we very often have a significant "halo" of cytoplasm around
>metaphases, which fluoresces slightly (with biotin-labelling, avidin
>development). I suspect that this may be due to cellular biotin picking
>up the label.

If guess if it's really cellular biotin that's been causing mischief in 
your FISH I suggest you try digoxigenin labelling. Unlike biotin which
is rather ubiquitous, digoxigenin (a steroid appended as a moiety to dUTP)
has only been found to date in the plant Digitalis purpurea. The
detection  I know for DIG is an ELISA based on alkaline phosphatase
which can also use a several fluorescent substrate systems. Boehringer
Mannheim (Germany) has been developing the DIG system. We have been using
their kit for our RFLPs for quite some time now and they work neatly. 
(I just hope I don't create an impression that I'm an agent of BM.)


R.P.Scott
Genet Lab
Plant Pathol Div
Intl Rice Res Inst
PO Box 933, 1099 Manila, Philippines
e-mail:  rscott at irri.cgnet.com



More information about the Methods mailing list