30 Microdissection of Chromosomes and Reverse FISH
403
nals were observed not only on the whole der(21) but on the normal chromosome 21 and on both chromosomes 11 (l1q14.2qter) as well.
A second example is presented in Fig. 3. Three copies of a small marker
chromosome from a renal cell carcinoma (for details see Glukhova et al.
1998) were isolated and amplified. The labeled products were hybridized
to normal metaphase spreads. Altogether, six distinct signals were observed, which were all localized on chromosome 7 (see Fig. 3). The question arising from these data was, whether all six chromosomal regions are
contained in each of the marker chromosomes, or if a diversity of different
marker chromosomes is present in the tumor, which contain different regions of chromosome 7. Hence, five single fragment microdissection experiments were carried out, where in five different amplification reactions
one single fragment was amplified. Reverse FISH with each of the DNA
libraries however, confirmed the initial results. All hybridizations exhibited the same fluorescent signals which were identical to the first result.
These investigations made clear, that indeed all collected marker chromosomes were identical.
Generation of painting probes by microdissection
Microdissection is not only an elegant way to characterize undefined chromosomes orchromosomal regions byreverse FISH,but can also be applied to
generate painting probes and partialpaintingprobes for use in forward FISH
experiments for characterizing chromosomal aberrations. In experienced
hands, the quality of painting probes generated by microdissection and
DOP-PCR is high and, they can even be used in mFISH experiments.
The principle of mFISH is based on the combinations of different fluorochromes for the labeling of single chromosome specific libraries (Fig. 4;
Schroock et al. 1996, Speicher et al. 1996).When applying this labeling strategy to all 24 different chromosome-specific paints, a spectral signature for
each chromosome is obtained. Five fluorochromes are required for unambiguous combinatorial labeling. With appropriate filter sets for the fluorochromes, digital images are captured which are further processed and
evaluated byadapted imaging systems. One elementaryfunction is to display
the 24 different chromosome classes of the human genome in 24 different
false colors according to the different combinations of fluorochromes
(Fig. 5). In this manner, complex interchromosomal exchanges can easily
be analyzed in one single FISH experiment. However, the mFISH technique
fails to detect intrachromosomal aberrations such as small deletions, which
inversion can. It has been shown in the literature that chromosome arm-
403
nals were observed not only on the whole der(21) but on the normal chromosome 21 and on both chromosomes 11 (l1q14.2qter) as well.
A second example is presented in Fig. 3. Three copies of a small marker
chromosome from a renal cell carcinoma (for details see Glukhova et al.
1998) were isolated and amplified. The labeled products were hybridized
to normal metaphase spreads. Altogether, six distinct signals were observed, which were all localized on chromosome 7 (see Fig. 3). The question arising from these data was, whether all six chromosomal regions are
contained in each of the marker chromosomes, or if a diversity of different
marker chromosomes is present in the tumor, which contain different regions of chromosome 7. Hence, five single fragment microdissection experiments were carried out, where in five different amplification reactions
one single fragment was amplified. Reverse FISH with each of the DNA
libraries however, confirmed the initial results. All hybridizations exhibited the same fluorescent signals which were identical to the first result.
These investigations made clear, that indeed all collected marker chromosomes were identical.
Generation of painting probes by microdissection
Microdissection is not only an elegant way to characterize undefined chromosomes orchromosomal regions byreverse FISH,but can also be applied to
generate painting probes and partialpaintingprobes for use in forward FISH
experiments for characterizing chromosomal aberrations. In experienced
hands, the quality of painting probes generated by microdissection and
DOP-PCR is high and, they can even be used in mFISH experiments.
The principle of mFISH is based on the combinations of different fluorochromes for the labeling of single chromosome specific libraries (Fig. 4;
Schroock et al. 1996, Speicher et al. 1996).When applying this labeling strategy to all 24 different chromosome-specific paints, a spectral signature for
each chromosome is obtained. Five fluorochromes are required for unambiguous combinatorial labeling. With appropriate filter sets for the fluorochromes, digital images are captured which are further processed and
evaluated byadapted imaging systems. One elementaryfunction is to display
the 24 different chromosome classes of the human genome in 24 different
false colors according to the different combinations of fluorochromes
(Fig. 5). In this manner, complex interchromosomal exchanges can easily
be analyzed in one single FISH experiment. However, the mFISH technique
fails to detect intrachromosomal aberrations such as small deletions, which
inversion can. It has been shown in the literature that chromosome arm-