Chapter 3
FISH on Chromosome Preparations of Peripheral Blood
THOMAS LIEHR and UWE CLAUSSEN
Introduction
Peripheral blood is the most frequently used tissue for chromosome studies, as it is easy to obtain, can simply be brought into short-term culture
and metaphase spreads can be prepared in high quality within a short
time. Such metaphase spreads, as well as the huge amounts of previously
superfluous interphase nuclei in cytogenetic preparations, are a material
very well suited to FISH analyses. All kinds of FISH probes can be used to
analyze metaphase spreads, while the interphase nuclei can actually be
analyzed successfully only by satellite and (sub-)telomere probes, plasmids, cosmids, BACs, YACs or PI-clones and not - at least not in routine-approaches - by whole or partial chromosome painting probes. However, the quality of metaphases is important in routine diagnostics, as well
as in research approaches (e.g. gene mapping). Thus, the preparation procedure of the chromosomes themselves is discussed in more detail in the
following.
The currently used method of cell culture and chromosome preparation is a result of the combination of many single step procedures introduced into the protocol by different researchers. An aliquot of peripheral
blood is added to culture medium, mixed with 10-20% fetal calf serum,
penicillin/streptomycin to avoid contaminating prokaryotic cell growth
and phytohaemagglutinin. The latter is a mitogen, stimulating in vitro
growth of T lymphocytes (Nowell 1960; Moorhead et al. 1960). After
72 h of incubation at 37°C/5% CO 2 , mimicking the conditions in human
veins, cells are harvested. The addition of colcemid (= deacetylmethylcolchicine) acts as a mitotic spindle inhibitor, so that a block is introduced
~ Thomas Liehr, Institut fur Humangenetik, Kollegiengasse 10, 'ena, 07743, Germany
(phone +49-3641-935533; fax +49-3641-935502; e-mail i8Iith@mti-n.mti.uni-jena.de)
Uwe Claussen, Institut fur Humangenetik, Kollegiengasse 10, 'ena, 07743, Germany
PROTOCOL
FISH on Chromosome Preparations of Peripheral Blood
THOMAS LIEHR and UWE CLAUSSEN
Introduction
Peripheral blood is the most frequently used tissue for chromosome studies, as it is easy to obtain, can simply be brought into short-term culture
and metaphase spreads can be prepared in high quality within a short
time. Such metaphase spreads, as well as the huge amounts of previously
superfluous interphase nuclei in cytogenetic preparations, are a material
very well suited to FISH analyses. All kinds of FISH probes can be used to
analyze metaphase spreads, while the interphase nuclei can actually be
analyzed successfully only by satellite and (sub-)telomere probes, plasmids, cosmids, BACs, YACs or PI-clones and not - at least not in routine-approaches - by whole or partial chromosome painting probes. However, the quality of metaphases is important in routine diagnostics, as well
as in research approaches (e.g. gene mapping). Thus, the preparation procedure of the chromosomes themselves is discussed in more detail in the
following.
The currently used method of cell culture and chromosome preparation is a result of the combination of many single step procedures introduced into the protocol by different researchers. An aliquot of peripheral
blood is added to culture medium, mixed with 10-20% fetal calf serum,
penicillin/streptomycin to avoid contaminating prokaryotic cell growth
and phytohaemagglutinin. The latter is a mitogen, stimulating in vitro
growth of T lymphocytes (Nowell 1960; Moorhead et al. 1960). After
72 h of incubation at 37°C/5% CO 2 , mimicking the conditions in human
veins, cells are harvested. The addition of colcemid (= deacetylmethylcolchicine) acts as a mitotic spindle inhibitor, so that a block is introduced
~ Thomas Liehr, Institut fur Humangenetik, Kollegiengasse 10, 'ena, 07743, Germany
(phone +49-3641-935533; fax +49-3641-935502; e-mail i8Iith@mti-n.mti.uni-jena.de)
Uwe Claussen, Institut fur Humangenetik, Kollegiengasse 10, 'ena, 07743, Germany
PROTOCOL