Chromosome Microdissection Using
During cytogenetic analysis there are frequently
complex chromosomal structural aberrations, which
are unidentifiable by chromosome banding analysis.
Fluorescence in situ hybridization (FISH) combined
with G-banding analysis (Xu and Wang, 1994) has
significantly improved the accuracy of chromosome
identification. However, the efficacy of FISH analysis
depends on the probes chosen, which in turn depend
on the G-banding result. It is very difficult to choose
the adequate probe(s) for FISH analysis when no clue
is available from banding analysis. Therefore, FISH
can serve as a tool for verification and confirmation.
The recently developed multicolor or spectral karyotyping
highly facilitates the identification of complex
interchromosomal structural abnormalities. However,
its application is limited to nonhomologous rearrangements
and its efficacy depends on the size of the
chromosome segment involved. Furthermore, it can
identify the chromosomes involved but not the specific
segment or breakpoints involved.
In contrast, the chromosome microdissection
approach provides a straightforward method for identifying
any chromosomal segment of unknown origin
by dissecting the segment of interest and isolating,
amplifying, fluorescence labeling, and reverse in situ
hybridizing DNA to normal metaphase spreads
(Ludecke et al.
, 1989; Bohlander et al.
, 1992; Carter et al.
, 1992; Meltzer et al.
, 1992; Ruano et al.
Telenius et al.
, 1992; Guan et al.
, 1993; Zhang et al.
1993). Once the components of the aberrant chromosome
are identified, conventional FISH with whole
chromosome painting probes can be applied to define
the relative position of the components in the marker
chromosome. This micro-FISH approach has been
applied successfully in the identification of ring chromosomes
(Xu et al.
, 1995, 1998), homogeneous staining
regions (Xu et al.
, 1996; Abeysinghe et al.
, 1999), and
double minutes (Sen et al.
, 1994). Thus, micro-FISH can
be used to identify not only the origin of the marker
chromosome, but also the regions and breakpoints.
However, in the reverse FISH the specificity of the
probe generated relies on the accurate recognition and
dissection of the specific target chromosome/segment
and on the precise extraction, amplification, and labeling
of the DNA with minimum background contamination.
The skills, procedures, and setup involved in
micro-FISH are too complicated to be established in the
routine clinical cytogenetic laboratory. This is the major
limitation to the application of reverse FISH by chromosome
microdissection. With a modified fixation
procedure (Xu et al.
, 1995), however, specimens can be
stored and shipped to experienced laboratories or diagnostic
centers. In addition to its clinical application,
chromosome microdissection can be used to generate
band-specific probes (Guan et al.
, 1993) that can be used
in establishing intrachromosomal spectral karyotyping.
A combination of interchromosomal spectral karyotyping
with intrachromosomal spectral banding analysis
would be ideal for chromosome analysis in neoplasia.
II. MATERIALS AND
Methyl alcohol (Fisher Scientific, #MD3017-4)
Potassian chloride solution 0.075N
Acetic acid, glacial ACS (Fisher Scientific, #A38-500)
Colcemid 10µl/ml in HBSS (GIBCO, #15210-040)
Trypsin (1:250) (GIBCO, #27250-018)
KaryoMax Giemsa stain (Lab Chem Inc., #2C 14840-7)
Hank's balanced salt solution ×1; without CaCl2
Buffer tablets "GURR" (GIBCO, #10582-013)
Deionized distilled (dd)water
T7 DNA polymerase (Sequenase, Version 2.0, USB
Topoisomerase 1 (Promega, #M2851)
Biotin-16-dUTP (Boehringer Mannheim, #1093070)
Taq DNA polymerase (Applied Biosystems, #4311816)
Universal primer 5'-CCGACTCGAGNNNNNNNATGTGG-3' (Telenius et al.
15-ml sterile polystyrene tubes (Corning, #43005)
Microneedle (Sutter Instrument Co., #BR100-15)
Coverslips (VWR, 24 × 60mm #48393-106)
Inverted microscope (Nikon DIAPHOT-TMD)
9-in. Pasteur glass pipettes (VCR, #53283-915) and
Micromanipulators (Narashige, Models MM-88 and
Micropipete puller (Narishige, Model PB-7)
Thermal cycler (MJ Research Inc., Model PTC-200)
Slide warmer (VWR)
Coplin jars (0.50-ml capacity)
Thin-walled microtubes (Laboratory Products Sales,
Microcon YM-100 columns (Millipore, #42413)
A. Preparation of Cells for Microdissection
This procedure is modified from those of Wang and
Federoff (1972) and Xu et al.
- Colcemid rnetaphase arresting solution: 10µg/ml
- Hypotonic solution: Prepare daily by mixing 0.4%
KCl (EM Science PX1405-1) (in dd H2O) and 0.4%
sodium citrate (EM Science PX0445-1) (in dd H2O)
in equal volume
- Fixation solution: Prepare daily by mixing methanol
(Mallinckrodt CAS 67-56-1) and glacial acetic acid
(Fisher Scientific A38-500 CAS 64-19-7) in a ratio of
- Trypsin solution: 0.625g trypsin (1:250; GIBCO
#27250-018) in 100ml dd H2O
- GURRS buffer solution: Dissolve one GURRS buffer
tablet (GIBCO 10582-013) in 1 liter dd H2O, adjust
pH to 6.8
- Giemsa staining solution: 2-3ml Giemsa (LabChem
Inc. LC14840-7) in 50ml GURRS buffer solution
B. Chromosome Microdissection Combinded
with FISH Analysis (Micro-FISH)
- Grow cells in cell culture medium supplemented
with 10% fetal bovine serum at 37°C in a 6% CO2 humidified incubator.
- Prepare chromosome metaphase spreads from a
proliferative cell population using colcemid (5-
10µl/ml) for metaphase arresting, hypotonic
treatment followed by fixation for chromosome
- Harvest cells either in situ or in suspension and then
drop on 24 × 60-mm #1.5 coverslips.
- Perform Giemsa-trypsin-G-banding analysis (GTG)
by standard procedure (Wang and Federoff, 1972)
for identification of the target chromosome or
chromosome segment for microdissection using an
The procedure of micro-FISH is performed according
to Guan et al.
(1993) with modification (Xu et al.
1. Chromosome Microdissection and
- 0.625% banding trypsin: Add 0.25g trypsin (1:250)
powder to 100ml dd H2O, mix well, aliquot to
2.0ml, and store at -20°C
- GURR buffer: Reconstitute with ddH2O (1 tablet/
1 liter), adjust pH to 6.8
- Collection buffer (5µl): 40mM Tris-HCl, pH 7.5,
20mM MgCl2, 50mM NaCl, 200µM of each dNTP,
1 unit Topo 1, and 5 pmol of universal primer
- PCR reaction mixture (50µl): 10mM Tris-HCl, pH
8.4, 2mM MgCl2, 50mM KCl, 0.1mg/ml gelatin,
200 µm of each dNTP, 50 pmol universal primer, and
2 units Taq DNA polymerase.
- T7 DNA polymerase working solution: Dilute T7 DNA
polymerase (13U/µl) with dilution buffer to
- Labeling solution (50 µl): 100 mM Tris-HCl, pH 8.4,
2mM MgCl2, 50mM dcl, 0.1mg/ml gelatin, 200µl
4dNTP, 50pmol universal primer, 20µM biotin-16-
dUTP, and 2 units Taq DNA polymerase.
- Whole chromosome painting probes obtained from
commercial laboratories (Vysis)
|FIGURE 1 Microdissection procedure in progress.
needle is aligned using a micromanipulator and
band or region is dissected. The needle
then used to pick up the
dissected piece (C) and
it into a collection tube.
- Prior to microdissection, fix cells in fixative solution
by three washes.
- Prepare metaphase spreads on 24 x 60-mm #1.5
coverslips and stain by GTG banding.
- Make microneedles with the micropipette puller
according to the manufacturer's manual and UV
treat overnight to rule out background DNA
- Perform microdissection on the target chromosome/
segment, which is identified by its specific
G-banding pattern, with glass microneedles
(Fig. 1) controlled by a Narashige micromanipulator
(Models MM-88 and MO-302) attached to an
- Transferr the dissected chromosome adherent to
the microneedle to a 0.2-ml microcentrifuge tube
containing 5 µl of collection buffer.
- Cover the collection buffer with 45 µl of mineral oil
and incubate at 37°C for 30min, followed by heat
soak at 94°C for 30min.
- Use a gene Amp PCR System 9600 (Perkin-Elmer
Atus) for DNA amplification.
- Perform an initial eight cycles of PCR (denaturation
at 94°C for lmin, anneling at 30°C for 2min,
and extension at 37°C for 2min) by adding approximately 0.3 units of T7 DNA polymerase
(Sequence Version 2.0, USB) at each cycle.
- Add 50µl of PCR reaction mixture to the tube.
- Heat the reaction to 95°C for 3 min followed by 35
cycles at 94°C for lmin, 56°C for lmin, 72°C for
2min, with a final extension at 72°C for 5 min.
2. Fluorescence Labeling of the PCR Product (Fig. 2)
|FIGURE 2 Diagram and flowchart of the micro-FISH
Once the whole chromosome or segment is
dissected, it is transferred
into a collection tube and PCR
amplified using random
primers. An aliquot of this reaction
is then reamplified by PCR with
the addition of
biotin/digoxigenin dUTP. The labeled PCR products
column purified for use as FISH probes.
3. Verification of the Specificity of the Dissected
- Label a 2-µl aliquot of the PCR product for 16 cycles
in a 50-µl secondary PCR with the same procedure as described earlier, except for the addition of
20 mM biotin-11-duTP (BMB).
- Purify the labeled PCR products using a Centricon
100 (Amicon) filter.
- Add 100µg of the probe to a 10-µl hybridization
mixture containing 55% formamide, 10% detran,
1 x SSC, and 5-10µg COT-1 DNA (BCL).
4. Identification of the Origin of the Dissected
- Grow the cells with the dissected chromosome/
segment on 22 x 22-mm coverslips in 35 x 10-mm
- Block the cells by treatment with colcemid (Gibco),
0.1µg/ml for 1 h, hypotonically treated, fixed, and
harvested using standard procedures.
- G-band the chromosomes following trypsin treatment
and stain with Giemsa stain.
- After GTG-banding and karyotyping, destain slides
with xylene, xylene/ethanol (1/1, v/v), and
methanol/acetic acid (3/1, v/v), refix in 3.7%
formaldhyde/phosphate-buffered saline (PBS), air
dry, and reuse for FISH as described by Klever et al. (1991) using the biotin-labeled probe generated
from the dissected chromosome/segment to verify
the specificity of the probe.
- Once the specificity of the probe is confirmed, the
probe can be hybridized back to normal metaphase
spreads to identify the origin of the dissected chromosome
5. Forward FISH Analysis to Identify the Relative
Position of the Components of the Dissected
- Analyze normal metaphase spreads by GTG
- Apply FISH analysis with the probe specific for
the dissected chromosome/segment to normal
metaphase spreads after GTG-banding analysis
using the procedure described in the previous
- Combine the result obtained from GTG banding
with that obtained from FISH analysis to identify
the origin or components of the chromosome/segments
- Apply FISH analysis with the commercially available
whole chromosome painting probes specific for each of the chromosome components, which are
identified by microdissection to the GTG-banded
metaphase spreads containing the dissected chromosome.
- Combining the G-banding and FISH results
obtained from each probe determines the relative
position of the components on the chromosome
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