Fine Mapping of Gene Ordering
by Elongated Chromosome Methods
Fluorescence in situ
hybridization (FISH) can be
used to localize specific DNA sequences on metaphase
chromosomes, interphase nuclei, and experimentally
extended DNA or chromatin fibers. Depending on the
hybridization target, FISH techniques show widely
different levels of DNA resolution (Table I). They
provide a rapid and accurate alternative to traditional
molecular approaches for long-range physical
mapping, including pulsed-field gel electrophoresis
and radiation hybrid mapping. One important application
has been the visual construction of contig maps
of regions of interest, such as disease gene loci or
chromosome breakpoints. Differential fluorescent
probe labeling allows the ordering of multiple DNA
sequences on the same chromosome. The DNA resolution
of standard metaphase FISH is in the 1- to 3-Mb
size range. Hybridization on less condensed interphase
nuclei increases the resolution to around 100 kb.
The average distance between two probe signals in the
three-dimensional nuclear space correlates with physical
distance up to 1-2Mb. This allows one to determine
the relative order and proximity of closely
juxtaposed sequences, which cannot be resolved on
metaphase chromosomes (Trask et al.
, 1989). Protocols
for stretching out DNA in solution (Bensimon et al.
1994) or the chromatin of interphase nuclei (Parra
and Windle, 1993; Haaf and Ward, 1994b; Heiskanen et al.
, 1994) have pushed the limits of resolution even
further. Hybridization to experimentally extended
DNA or chromatin fibers provides a mapping
power in the order of 1-10 kb. The maximum distance
possible for clone ordering may be up to several
megabases; however, the average size of intact fibers
is considerably smaller. Most fiber FISH techniques
are informative over genomic distances from 100 to
Mechanically stretched or elongated chromosomes
fill the resolution gap between metaphase FISH and
fiber FISH, allowing the rapid and straightforward
ordering and localization of clones along the length of
an entire chromosome with a 100- to 200-kb resolution.
Elongated chromosomes for high-resolution FISH
are prepared by centrifuging colcemid-arrested
metaphase spreads in hypotonic solution to microscope
glass slides. The mechanical forces generated
during cytocentrifugation result in mechanical stretching
of chromosomes to 5 to 20 times their normal
length due to their elastic consistency under moist
conditions (Haaf and Ward, 1994a,b). Elongated chromosome
FISH can be applied to large numbers of
probes and provides a more than 10-fold increase in
DNA resolution compared to standard metaphase
mapping. This makes it possible to create 100-kb
physical maps of a chromosome(s) or chromosome
region(s) of interest in relatively short time (Laan et al.
Although various genome projects have provided
very high-resolution physical maps of human and
important animal genomes, FISH is still an exceptionally
versatile mapping tool. For example, working
with duplicated or repetitive sequences at the molecular
level can be extremely tedious and, therefore,
accurate mapping and sequencing of these regions
remain tremendous tasks in the postgenomic era.
In addition, FISH has important applications for
mapping chromosome regions that have been deleted,
amplified, and/or rearranged in human pathology or
evolution. Of course, FISH techniques are useful not
only for mapping the human genome. They can be adapted easily for the study of map-poor genomes
from primitive vertebrates to humans.
II. MATERIALS AND
All reagents for elongated chromosome preparation
are standard laboratory chemicals that can be obtained
from many different suppliers, i.e., NaCl (Sigma-
Aldrich, Cat.No. S7653), KCl (Sigma-Aldrich, Cat.No.
(Sigma-Aldrich, Cat.No. S9638),
(Sigma-Aldrich, Cat.No. P5655), HEPES dry
powder (Sigma-Aldrich, Cat.No. H9897), glycerol
99.5% ACS reagent (Sigma-Aldrich, Cat.No. G7893),
O (Sigma-Aldrich, Cat.No. C3881), MgCl2·
O (Sigma-Aldrich, Cat.No. M0250), acetic acid
(VWR, Cat.No. 1.00058.1000), methanol (VWR, Cat.No.
1.06008.1000), acetone (VWR, Cat.No. 1.59005.0500),
Biocoll (Ficoll) separating solution (Biochrom, Cat.No 2
6115), and colcemid (Roche, Cat.No. 295892).
The cytocentrifuge (Cytospin 3) and the filter blots
are from Thermo Shandon Inc.
A. Preparation of Elongated Chromosomes for
The following protocol describes the preparation of
elongated chromosomes from human lymphoblastoid
or fibroblast cells (Haaf and Ward, 1994a,b). This technique
can be adapted for other cell substrates, producing
suitable targets for high-resolution FISH
- Phosphate-buffered saline (PBS): 136mM NaCl,
2mM KCl, 10.6mM Na2HPO4, and 1.5 mM KH2PO4,
pH 7.3. To make 10× PBS stock solution, weigh 79.5g
NaCl, 1.5 g KCl, 15 g Na2HPO4, and 2 g KH2PO4. After
dissolving in distilled water, complete to 1000ml, pH,
and autoclave. Store at room temperature. To make 1× PBS, dilute stock solution 1 : 10 with distilled water and
use within 1 day.
- Hypotonic solution (for cell swelling): 10mM HEPES, 30mM glycerol, 1 mM CaCl2, and 0.8mM MgCI2. To make 1000 ml, add 2.4g HEPES, 2.2ml anhydrous
glycerol, 147mg CaCl2·2H2O, and 163mg
MgCl2·6H2O. After dissolving, complete to 1000ml
with distilled water, pH, and filter if necessary. Store
at 4°C and use within 1 week.
- Carnoy's fixative: Mix one part acetic acid and
three parts methanol. Store at -20°C and use within 1
B. Preparation of Elongated Chromosomes
for Combined Protein Immunofluorescence
- After harvesting a mitotically dividing cell
culture(s), wash the cells one time in 1× PBS and resuspend
the cell pellet in hypotonic solution to a concentration
of 103 to 104 cells/ml. Incubate the cells at
ambient temperature for 5-10min.
- Centrifuge 0.5-ml aliquots of this hypotonic
cell suspension onto microscope slides at 1000rpm
for 4min using a Shandon cytospin. Specifically, place
the cell suspension in the cytobuckets of the centrifuge,
the bases of which are formed by a filter blot and a
glass slide (cleaned with ethanol). The whole structure
is held together by a clip. During centrifugation the
suspension fluid is absorbed by the filter blot and the
cells become attached to the glass slide. After cytocentrifugation,
the slide should carry a visible monolayer
of cells in a designated area about 5 mm in diameter.
Use a diamond pencil to mark the back of the
slide to indicate the area on which the cells are
- Fix the slides for 30min at -20°C in methanol
and then overnight at -20°C in Carnoy's fixative. Airdried
slides stored at 4°C can be kept for months
Select slides suitable for FISH mapping using a
phase-contrast microscope at low magnification (10 to
20× lens without oil). A higher density of cells per slide
results in poor chromosome stretching and cytoplasmic
background ("dirt"). A lower density of cells
results in disruption of metaphase spreads and
individual chromosomes. Chromosomes extended
to 5 to 20 times their normal length but still intact
are most useful for high-resolution chromosome
mapping. Good slides should contain 3-10 metaphase
spreads with stretched chromosomes without broken
arms. If less than half of the slides obtained after
cytocentrifugation meet these criteria, the cell density
in the hypotonic solution was either too high or too
|FIGURE 1 (A and B) Two-color FISH with two
(<1Mb) YACs from the
5q34-q35 region to chromosomes mechanically
stretched to 5-10 times their normal length. (C)
of centromeric proteins (green)
and a-satellite DNA (red) on chromosomes
stretched to 10-20 times their normal length.
proteins were detected by indirect
antiserum and FITC-labeled antihuman IgG.
DNA was hybridized in situ with a
digoxigenated human α-satellite
and detected with rhodamine-conjugated
antibody. Bar: 10 µm.
- Standard FISH protocols are used for mapping
cloned DNA sequences such as cosmids, BACs, or
YACs to acid-fixed elongated chromosomes (Haaf,
2000). Usually one probe is detected with a green fluorescent
dye, a second probe with a red dye, and the
chromosomes are counterstained in blue with DAPI
(Figs. 1A and 1B). However, using modern FISH workstations
for M-FISH or spectral karyotype analysis
(SKY), many more DNA targets can be distinguished
in different colors.
- Determine the relative order of two hybridized
clones that are <1Mb apart on at least five elongated
chromosomes. Because mechanically stretched chromosomes
can be highly deformed, not all hybridized
chromosome copies may be informative. If the order is
not consistent on all five chromosomes, analyze five
additional chromosome copies. In most cases, one
slide should be sufficient for ordering probes that are
100-200 kb apart.
The combined application of protein staining by
immunofluorescent techniques and DNA staining by
FISH provides a powerful tool for mapping chromosomal
proteins to specific DNA sequences. Steps
IV. COMMENTS AND PITFALLS
- Harvest mitotic cells and centrifuge them onto
slides as described earlier.
- Fix the slides in methanol at -20°C for 30 min and
then immerse in ice-cold acetone for a few seconds.
After brief air drying, process the preparations for
immunofluorescence staining. Because most protein
antigens are not very stable, it is not recommened to
store the slides before use.
- Perform immunofluorescent staining of a protein
of interest, i.e., as an example only, labeling of centromeric
proteins (CENPs) by specific anti-CENP antibodies
(Fig. 1C). Standard immunofluorescent
protocols are used for incubation of the slides with
primary and fluorescent-labeled secondary antibodies
(Haaf, 1995). However, do not counterstain the preparations
- Immediately after immunofluorescent staining,
refix the preparations by incubating them in Carnoy's
fixative overnight at -20°C and then hybridize them
with a DNA probe(s) of interest.
- The most suitable source of mitotic cells are longterm in vitro cell cultures such as EBV-transformed
lymphoblasts, fibroblasts, or established cell lines.
Usually, 10µg/ml colcemid (Roche) is added to the
culture medium 10-15 min before cell harvest to arrest
cells in metaphase. Whole blood cultures cannot be
used as a cell substrate because the erythrocytes are
not destroyed by the hypotonic treatment required for
elongated chromosome preparation. Therefore, it is
necessary to separate white blood cells from red cells
by density gradient centrifugation (in Biocoll/Ficoll
separating solution) prior to short-term lymphocyte
- Elongated chromosomes constitute a compromise
between different demands. The mechanical
stretching procedure inevitably impairs chromosome
morphology. The chromosomes are deformed and
cannot be banded; however, they provide a greater
DNA resolution and allow the ordering of probes
from the short-arm to the long-arm telomere. Usually,
better chromosome morphology goes along with poor
stretching and vice versa.
- Chromosome elongation is not uniform over the
entire length of a stretched chromosome and the same
metaphase spread may contain both well-extended
and poorly stretched chromosomes. Therefore it is
very difficult to correlate the micrometer distance
between two mapped loci or the relative distance from
the short arm telomere (Flpter value) with physical
distance in kilobases. Some regions, i.e., telomeres,
which are usually attached to the surface of the glass
slide, are more resistant to stretching than others.
- Carnoy's fixation of immunofluorescent preparations
and subsequent FISH do not grossly interfere
with the fluorecent-labeled protein antigen-DNA
complex. Although the intensity of the fluorescent
protein signal is reduced to a certain extent, it is still
visible after the relatively harsh in situ hybridization
procedure. This allows one to analyze the results of
protein immunofluorescence and DNA in situ hybridization simultaneously on the same elongated
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