8
EIGIL KJELDSEN and STEEN K0LVRAA
CYTOPLASM
RNA viruses
e.g. retrovirus. parvovirus
RNA transcripts from
DNA viruses
Gene expression
- endogenous
- exogeneous
e.g. RNA of transfected DNA
Viral RNA
Mitochondrial
nucleic acids
mRNA
Nuclear or
'-+'*"-~ chromosomal DNA
~'H-~Viral RNA
HPV
EBV
Adenovirus
CMV
ChromosomeslDNA fibers
Centromeric sequences
Telomeric sequences
Individual genesJ10ci
Fig. 1. Targets for in situ techniques
Technical elements
The specific annealing of probe to chromosomal or nuclear DNA is schematically illustrated in Fig. 2. The target DNA, e.g. chromosomes or nuclei,
is first bound to the surface of glass slides. The DNA is then denatured by
heating either before or after applying a special mixture that allows for
annealing or hybridization of the single stranded probe to complementary
sequences in the target DNA. Hybridization is usually performed overnight at a specific temperature which, in combination with the hybridization mixture determines the specificity of the annealing. Surplus probe is
finally washed off in a stringent wash and the specifically annealed probefragments can subsequently be demonstrated by fluorescence microscopy.
The standard FISH protocol can be described as having six steps, each
of which is crucial for obtaining a successful FISH-result (practical details
can be found in Levy 1995, Choo 1994, Wilkinson 1992):
1. Sample preparation (source, fixation and unmasking)
2. Probe preparation and labeling
3. Denaturation of probe and sample
4. Hybridization of probe to sample (annealing)
5. Post-hybridization washing
6. Detection