1 FISH Techniques, FISH Probes and Their Applications in Medicine and Biology - An Overview
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hensive characterization of changes in the relative copy number of chromosomal material (Kallioniemi et al. 1992). The technique can also be
used when no viable cells are available and has even been used to characterize the whole genome from a single cell. However, with CGH it is not
possible to identify balanced structural aberrations.
Genomic DNA is purified from the tissue or cell line of interest. This
DNA is then labeled by nick-translation using nucleotides coupled to, for
example FITC. Control DNA is also labeled by nick-translation using nucleotides coupled to rhodamine. Equal amounts ofthese two DNA samples
are then allowed to competitively hybridize onto a normal metaphase
spread of human chromosomes. The red/green color ratio obtained after
hybridization indicates either a relative excess or deficiency of target DNA
(relative to the control) or equality of hybridization. The color-ratio analysis is generated by specialized software, which analyses images generated
by a CCD-camera, coupled to an epifluorescence microscope.
Analytical quality control is a major problem in CGH and perhaps
more important here than in conventional FISH. This is due to the
fact that the hybridization is analyzed quantitatively. In an effort to overcome this problem a new CGH modification, four-color CGH, was recently
developed (Karhu et aI., 1999). The modification provides an internal standard in every hybridization which helps in identifying inconsistently hybridized chromosomal regions and when using a special second reference
DNA, the dynamic range of hybridization can also be standardized.
The CGH technique also has sensitivity and resolution limitations. For
deletion detection the size of the deletion seems to be 5-10 Mb whereas for
gain detection, the amplicon size can be 100-200 kb.
DNA Chip technology
A way to improve the resolution of CGH is to artificially create microarrays of individually or appropriately pooled DNA clones or custom
synthesized oligonucleotides and use these as the reporting genome instead of the highly condensed metaphase chromosomes (Lichter et al.
1997). In principle, one can choose the CGH resolution that is needed
by selecting appropriately spaced DNA sequences in the microarrays.
The presence or absence of these sequences in the genome of a patient
can then be visualized by using DNA from the patient as a fluorescent
probe that is hybridized together with equimolar control genomic
DNA to these fixed sequences. The red/green color ratio obtained after
hybridization indicates either a relative excess or deficiency of target
23
hensive characterization of changes in the relative copy number of chromosomal material (Kallioniemi et al. 1992). The technique can also be
used when no viable cells are available and has even been used to characterize the whole genome from a single cell. However, with CGH it is not
possible to identify balanced structural aberrations.
Genomic DNA is purified from the tissue or cell line of interest. This
DNA is then labeled by nick-translation using nucleotides coupled to, for
example FITC. Control DNA is also labeled by nick-translation using nucleotides coupled to rhodamine. Equal amounts ofthese two DNA samples
are then allowed to competitively hybridize onto a normal metaphase
spread of human chromosomes. The red/green color ratio obtained after
hybridization indicates either a relative excess or deficiency of target DNA
(relative to the control) or equality of hybridization. The color-ratio analysis is generated by specialized software, which analyses images generated
by a CCD-camera, coupled to an epifluorescence microscope.
Analytical quality control is a major problem in CGH and perhaps
more important here than in conventional FISH. This is due to the
fact that the hybridization is analyzed quantitatively. In an effort to overcome this problem a new CGH modification, four-color CGH, was recently
developed (Karhu et aI., 1999). The modification provides an internal standard in every hybridization which helps in identifying inconsistently hybridized chromosomal regions and when using a special second reference
DNA, the dynamic range of hybridization can also be standardized.
The CGH technique also has sensitivity and resolution limitations. For
deletion detection the size of the deletion seems to be 5-10 Mb whereas for
gain detection, the amplicon size can be 100-200 kb.
DNA Chip technology
A way to improve the resolution of CGH is to artificially create microarrays of individually or appropriately pooled DNA clones or custom
synthesized oligonucleotides and use these as the reporting genome instead of the highly condensed metaphase chromosomes (Lichter et al.
1997). In principle, one can choose the CGH resolution that is needed
by selecting appropriately spaced DNA sequences in the microarrays.
The presence or absence of these sequences in the genome of a patient
can then be visualized by using DNA from the patient as a fluorescent
probe that is hybridized together with equimolar control genomic
DNA to these fixed sequences. The red/green color ratio obtained after
hybridization indicates either a relative excess or deficiency of target