22
EIGIL KJELDSEN and STEEN K0LVRAA
rule of thumb is that if)70% ofthe nuclei falls into one diagnostic category,
it can be diagnosed as such (Shaffer et al. 1997, Liehr et al. 1996).
Fiber-FISH
The resolution of FISH for metaphase chromosomes has been assessed to
be about 3 Mb which corresponds to a smaller band seen in G-or Qbanded chromosomes making metaphase FISH suited for only coarse
mapping of DNA clones. Thus, within a band, clone ordering by FISH
to metaphase chromosomes is virtually impossible.
Use of a more relaxed state of the chromatin as in interphase nuclei,
provides a dramatic increase in resolution. Interphase FISH resolution has
been estimated to be around 100 kb (Lawrence et al. 1990, Trask et al.
1991). The genomic resolution is limited by the spatial resolution of
the microscope and determined by the chromatin condensation state.
To improve FISH resolution either high-resolution microscopy (Moers
et al. 1996) or further decondensation of chromatin has been utilized.
As high-resolution microscopy is a rather expensive way of improving resolution power, a more practical approach is to use decondensed chromatin and conventional epi-fluorescence microscopy which can give resolutions up to the theoretical possible (Raap et al. 1996). Several strategies
have been pursued to achieve decondensed chromatin in the form of
naked DNA fibers and are collectively referred to as Fiber-FISH. For example, SDS-extraction of mammalian cell nuclei immobilized on glass
slides (Para and Windle 1993) or extraction conditions adapted from
the halo-preparation technique (Wiegant et al. 1992) can produce intact
stretches of several hundred kilobase pairs up to over 1 Mb of intact naked
DNA fibers at sufficient density.
Early Fiber-FISH results showed that the theoretical resolution could
be achieved in practice. A 1 kb oflinear DNA measures 0.34 Jlm in length
and optical resolution is 0.2-0.3 Jlm dependent on objective magnification
and numerical aperture. The detection sensitivity limit has been found to
be as low as 200 bp (Florijn et al. 1996).
Comparative genomic hybridization (CGH)
Comparative genomic hybridization (CGH) analysis is another powerful
refinement of the standard FISH technique, which can be used to screen
the whole genome from tissue samples or cell lines providing a compre-
EIGIL KJELDSEN and STEEN K0LVRAA
rule of thumb is that if)70% ofthe nuclei falls into one diagnostic category,
it can be diagnosed as such (Shaffer et al. 1997, Liehr et al. 1996).
Fiber-FISH
The resolution of FISH for metaphase chromosomes has been assessed to
be about 3 Mb which corresponds to a smaller band seen in G-or Qbanded chromosomes making metaphase FISH suited for only coarse
mapping of DNA clones. Thus, within a band, clone ordering by FISH
to metaphase chromosomes is virtually impossible.
Use of a more relaxed state of the chromatin as in interphase nuclei,
provides a dramatic increase in resolution. Interphase FISH resolution has
been estimated to be around 100 kb (Lawrence et al. 1990, Trask et al.
1991). The genomic resolution is limited by the spatial resolution of
the microscope and determined by the chromatin condensation state.
To improve FISH resolution either high-resolution microscopy (Moers
et al. 1996) or further decondensation of chromatin has been utilized.
As high-resolution microscopy is a rather expensive way of improving resolution power, a more practical approach is to use decondensed chromatin and conventional epi-fluorescence microscopy which can give resolutions up to the theoretical possible (Raap et al. 1996). Several strategies
have been pursued to achieve decondensed chromatin in the form of
naked DNA fibers and are collectively referred to as Fiber-FISH. For example, SDS-extraction of mammalian cell nuclei immobilized on glass
slides (Para and Windle 1993) or extraction conditions adapted from
the halo-preparation technique (Wiegant et al. 1992) can produce intact
stretches of several hundred kilobase pairs up to over 1 Mb of intact naked
DNA fibers at sufficient density.
Early Fiber-FISH results showed that the theoretical resolution could
be achieved in practice. A 1 kb oflinear DNA measures 0.34 Jlm in length
and optical resolution is 0.2-0.3 Jlm dependent on objective magnification
and numerical aperture. The detection sensitivity limit has been found to
be as low as 200 bp (Florijn et al. 1996).
Comparative genomic hybridization (CGH)
Comparative genomic hybridization (CGH) analysis is another powerful
refinement of the standard FISH technique, which can be used to screen
the whole genome from tissue samples or cell lines providing a compre-