Towards unlimited colors for fluorescence in-situ hybridization (FISH).
We describe a FISH protocol that allows rehybridization of complex DNA probes up to four times to the same specimen. This strategy, which we termed ReFISH, opens a wide range of new applications to conventional band pass filter epifluorescence microscopy. These include M-FISH karyotyping and cross-species color banding that emulate multiplex probe sets labeled with up to 12 fluorochromes in sequential hybridizations to the same specimen. We designed a human 24-color karyotyping probe set in combination with a 29-color cross-species color banding probe set using gibbon painting probes. Applying the ReFISH principle, 53 painting probes on individual metaphases were discriminated. This allowed simultaneous screening for inter- and intrachromosomal rearrangements on normal human diploid cells, a HeLa derived cell line, and highly rearranged gibbon chromosomes. Furthermore, the present ReFISH experiments successfully combine 24-color FISH with laser scanning confocal microscopy to study the 3D organization of all 46 human chromosome territories in individual interphase cell nuclei.
Müller
Stefan
S
Institute of Anthropology and Human Genetics, University of Munich (LMU), Germany.
Neusser
Michaela
M
Wienberg
Johannes
J
D000818:Animals; D002460:Cell Line; D002871:Chromosome Banding; D020223:Chromosome Painting / Q000379:methods*; D003116:Color; D006367:HeLa Cells; D006801:Humans; D006922:Hylobates / Q000235:genetics; D007621:Karyotyping; D008214:Lymphocytes / Q000648:ultrastructure; D018613:Microscopy, Confocal; D013045:Species Specificity