38
EIGIL KJELDSEN and STEEN K0LVRAA
in vitro fusion of human cells with cells from other species, various molecular techniques and in situ hybridization.
Since ISH was used for the first time in 1981 for localization of a single
gene on metaphase chromosomes, the technique has proven to be an invaluable gene-mapping tool (Heiskanen et al. 1996). Further refinements
of the use of FISH as a gene-mapping tool has shown that it can be applied
to interphase nuclei, mechanically stretched chromosomes (Laan et al.
1995) or DNA fibers (Florijn et al. 1996) to order DNA sequences or genes
with increasingly greater resolution. Now individual genes can be mapped
as soon as they are cloned.
To determine the chromosome to which a probe has hybridized, a technique for chromosome identification compatible with CISS hybridization
has been developed (Klever et al. 1991). It is possible to do Giemsa banding
after hybridization but this process requires the relocalization of specific
metaphase spreads. Another strategy involves cohybridization with a differently labeled probe (or probe set). Alu DNA BLUR clone has been used
because it can give a banding pattern resembling R banding (Baldini and
Ward 1991). For simultaneous hybridization strategies, it is important to
have different labeling procedures that result in comparable fluorescent
signals.
The use of Fiber-FISH has contributed significantly to positional cloning efforts by facilitating physical mapping of probes from the chromosome region of interest by rapid and direct visualization of clone order,
and sizes of gaps and overlaps (Heng et al. 1992, Haaf and Ward 1994,
Michalet et al. 1997). Even copy numbers of moderately repeated sequences (Shiels et al. 1997) as well as viral integration (Lestou et al.
1996) have been assessed by Fiber-FISH.
Fiber-FISH has proven to be a valuable tool also in contig map construction. The technique also provides a rapid and informative means to
study gene rearrangements, because once a contig map of a well-covered
disease region has been made, it is a relatively simple task to create a FiberFISH color bar code by selecting adequately spaced and/or overlapping
clones and proper color assignment of individual DNAs. Gene rearrangements will then be apparent as a disruption of the wild-type bar code
thereby easily characterizing either deletion- or translocation breakpoints
scattering several hundreds of kilobases at a kilobase resolution (Vaandrager et al. 1996). The ability by Fiber-FISH to scan large genomic regions
for gene-rearrangements in a single FISH experiment makes such an approach advantageous over Southern hybridization and PCR-amplification
techniques.
EIGIL KJELDSEN and STEEN K0LVRAA
in vitro fusion of human cells with cells from other species, various molecular techniques and in situ hybridization.
Since ISH was used for the first time in 1981 for localization of a single
gene on metaphase chromosomes, the technique has proven to be an invaluable gene-mapping tool (Heiskanen et al. 1996). Further refinements
of the use of FISH as a gene-mapping tool has shown that it can be applied
to interphase nuclei, mechanically stretched chromosomes (Laan et al.
1995) or DNA fibers (Florijn et al. 1996) to order DNA sequences or genes
with increasingly greater resolution. Now individual genes can be mapped
as soon as they are cloned.
To determine the chromosome to which a probe has hybridized, a technique for chromosome identification compatible with CISS hybridization
has been developed (Klever et al. 1991). It is possible to do Giemsa banding
after hybridization but this process requires the relocalization of specific
metaphase spreads. Another strategy involves cohybridization with a differently labeled probe (or probe set). Alu DNA BLUR clone has been used
because it can give a banding pattern resembling R banding (Baldini and
Ward 1991). For simultaneous hybridization strategies, it is important to
have different labeling procedures that result in comparable fluorescent
signals.
The use of Fiber-FISH has contributed significantly to positional cloning efforts by facilitating physical mapping of probes from the chromosome region of interest by rapid and direct visualization of clone order,
and sizes of gaps and overlaps (Heng et al. 1992, Haaf and Ward 1994,
Michalet et al. 1997). Even copy numbers of moderately repeated sequences (Shiels et al. 1997) as well as viral integration (Lestou et al.
1996) have been assessed by Fiber-FISH.
Fiber-FISH has proven to be a valuable tool also in contig map construction. The technique also provides a rapid and informative means to
study gene rearrangements, because once a contig map of a well-covered
disease region has been made, it is a relatively simple task to create a FiberFISH color bar code by selecting adequately spaced and/or overlapping
clones and proper color assignment of individual DNAs. Gene rearrangements will then be apparent as a disruption of the wild-type bar code
thereby easily characterizing either deletion- or translocation breakpoints
scattering several hundreds of kilobases at a kilobase resolution (Vaandrager et al. 1996). The ability by Fiber-FISH to scan large genomic regions
for gene-rearrangements in a single FISH experiment makes such an approach advantageous over Southern hybridization and PCR-amplification
techniques.