12 Chromosome Analysis by Fluorescence in situ Hybridization
169
ogy: Hybridization can be performed on routine clinical material (Manuelidis 1985; Hopman et al. 1988; Anastasi et al. 1991; Bentz et al. 1993) as
well as preparations with well-preserved three-dimensional morphology
(Zirbel et al. 1993).
• Probes: A variety of probe types that can be categorized according to the
complexitiy of their target sequences are used for FISH. Clusters of chromosome-specific repeated sequences are detected by alphoid or satellite
probes. Composite probes allow specific "painting" (Cremer et al. 1988;
Lichteret al. 1988; Pinkelet al1988) of chromosomes or chromosomal
regions. Other probes such as most genomic DNA fragments cloned in
plasmid-, phage-, cosmid-, P1 or yeast artificial chromosome vectors detect unique loci in the genome. However, for the selection of probes from
this category, the size of the targeted DNA sequence must be taken into
consideration, as this correlates fairly well with the hybridization efficiency. With cosmids (25 to 40 kb of cloned insert) signals ofboth chromosome homologs are usually visualized in about 90% of cells. In contrast, probes targeting less than 5 kb of single copy sequences detect a
maximum of 50% of the complementary sequences (Joos et al. 1994).
These estimates refer to hybridizations performed on routine cytogenetic preparations.
• Probe labelling: The nucleic acid probes are labeled by the introduction
of specific reporter groups ( e.g. biotin, digoxigenin or FITC). In general,
Fig. 2. Analysis of deletions of the retinoblastoma gene (Rb-a) using phage clones spanning
the whole 200 kb of the retinoblastoma gene: A Metaphase spread of a healthy pro band counterstained with DAPI showing specific Rb-1 hybridization signals detected via FITC (yellow).
Painting of chromosomes 13 was achieved by cohybridization of chromosome-specific library
DNA detected via rhodamine (red). Cross-hybridizations oflibrary DNA occurs on the short
arms of other D- and G- group chromosomes. B Lymphocyte interphase nuclei of a patient
with chronic lymphocytic leukemia ( CLL) after hybridization with Rb-1 (green) and c518 that
was used as a control for hybridization efficiency (red}, and counterstain with DAPI. Three
cells show a Rb-1 deletion ( only one green signal}, whereas one cell has two copies of Rb-1
(two green signals). Note the two c518 signals (red) in each nucleus indicating a high hybridization efficiency. C Blood smear of the same patient after hybridization with Rb-1 and c518.
Three round nuclei and one lobulated nucleus likely to represent three lymphocytes and one
granulocyte, respectively, are visualized by DAPI counterstain. The granulocyte has two Rb-1
copies (two green signals}, whereas the other cells show an Rb-1 deletion (only one green
signal). The high efficiency is shown by two c518 signasl (red) per cell. D Metaphase spread
and interphase nuclei of the same patient counterstained with DAPI. While the metaphase
shows Rb-1 signals on each chromosome 13 homolog, both interphase nuclei exhibit adeletion of one Rb-1 copy (only one signal per cell). Reprinted from Stilgenbauer et al. 1993,
Saunders Company
169
ogy: Hybridization can be performed on routine clinical material (Manuelidis 1985; Hopman et al. 1988; Anastasi et al. 1991; Bentz et al. 1993) as
well as preparations with well-preserved three-dimensional morphology
(Zirbel et al. 1993).
• Probes: A variety of probe types that can be categorized according to the
complexitiy of their target sequences are used for FISH. Clusters of chromosome-specific repeated sequences are detected by alphoid or satellite
probes. Composite probes allow specific "painting" (Cremer et al. 1988;
Lichteret al. 1988; Pinkelet al1988) of chromosomes or chromosomal
regions. Other probes such as most genomic DNA fragments cloned in
plasmid-, phage-, cosmid-, P1 or yeast artificial chromosome vectors detect unique loci in the genome. However, for the selection of probes from
this category, the size of the targeted DNA sequence must be taken into
consideration, as this correlates fairly well with the hybridization efficiency. With cosmids (25 to 40 kb of cloned insert) signals ofboth chromosome homologs are usually visualized in about 90% of cells. In contrast, probes targeting less than 5 kb of single copy sequences detect a
maximum of 50% of the complementary sequences (Joos et al. 1994).
These estimates refer to hybridizations performed on routine cytogenetic preparations.
• Probe labelling: The nucleic acid probes are labeled by the introduction
of specific reporter groups ( e.g. biotin, digoxigenin or FITC). In general,
Fig. 2. Analysis of deletions of the retinoblastoma gene (Rb-a) using phage clones spanning
the whole 200 kb of the retinoblastoma gene: A Metaphase spread of a healthy pro band counterstained with DAPI showing specific Rb-1 hybridization signals detected via FITC (yellow).
Painting of chromosomes 13 was achieved by cohybridization of chromosome-specific library
DNA detected via rhodamine (red). Cross-hybridizations oflibrary DNA occurs on the short
arms of other D- and G- group chromosomes. B Lymphocyte interphase nuclei of a patient
with chronic lymphocytic leukemia ( CLL) after hybridization with Rb-1 (green) and c518 that
was used as a control for hybridization efficiency (red}, and counterstain with DAPI. Three
cells show a Rb-1 deletion ( only one green signal}, whereas one cell has two copies of Rb-1
(two green signals). Note the two c518 signals (red) in each nucleus indicating a high hybridization efficiency. C Blood smear of the same patient after hybridization with Rb-1 and c518.
Three round nuclei and one lobulated nucleus likely to represent three lymphocytes and one
granulocyte, respectively, are visualized by DAPI counterstain. The granulocyte has two Rb-1
copies (two green signals}, whereas the other cells show an Rb-1 deletion (only one green
signal). The high efficiency is shown by two c518 signasl (red) per cell. D Metaphase spread
and interphase nuclei of the same patient counterstained with DAPI. While the metaphase
shows Rb-1 signals on each chromosome 13 homolog, both interphase nuclei exhibit adeletion of one Rb-1 copy (only one signal per cell). Reprinted from Stilgenbauer et al. 1993,
Saunders Company