General Introduction to In Situ Hybridization

5
74
Procedures for In Situ Hybridization to Chromosomes, Cells, and Tissue Sections
ISH to whole chromosomes
6 Analyze aliquots of the reaction mixtures
on an agarose gel by performing
the following steps:
Mix the aliquots with gel loading
buffer (0.25% bromophenol blue and
30% glycerol).
Load samples on a minigel. As a size
marker use a 1 kb ladder or other
suitable size standard.
Run the gel in 1x TBE buffer (89 mM
Tris base, 89 mM boric acid, 2 mM
EDTA; pH 8.0) for 30 min at 100
volts.
7 Stain the gel with ethidium bromide and
photograph. In the reaction samples, you
should see a smear of DNA, ranging in
size from about 200 bp to 2000 bp. No
smear should be visible in the negative
control.
8 Collect the amplified DNA products
from the reaction mix by using the High
Pure PCR Product Purification Kit, see
page 50.
9 Label the amplified DNA by standard
nick translation procedures (Lichter and
Cremer; Lichter et al., 1994).
II. Comparative genomic
hybridization (CGH)
1 Prepare slides of normal metaphase
chromosomes as described in the
literature (Lichter and Cremer; Lichter
et al., 1990).
2 Prepare the following solutions:
3 M sodium acetate, pH 5.2.
Deionized formamide (molecular
biology grade): For deionization, use
ion exchange resin. The conductivity
of the formamide should be below
100 µSiemens.
Hybridization buffer (4 x SSC, 20%
dextran sulfate): Prepare 20 x SSC.
Carefully dissolve dextran sulfate in
water to make a 50% dextran sulfate
solution. Autoclave the dextran sulfate
solution or filter it through a
nitrocellulose filter. Mix 200 µl of
20 x SSC, 400 µl of 50% dextran sulfate
and 400 µl of double-distilled
water. Store the hybridization buffer
at 4°C until you use it.
3 To prepare probe DNA, combine 1 µg
of each labeled test and control DNA
with 50–100 µg of human Cot-1 DNA.
4 Co-precipitate the probe DNAs in each
tube by adding 1/20 volume of 3 M
sodium acetate and 2.5 volumes of
100% ethanol. Mix well and incubate at
–70°C for 30 min.
5 Spin the precipitated DNA in a
microcentrifuge at 12,000 rpm for 10
min at 4°C. Discard the supernatant and
wash the pellet with 500 µl of 70%
ethanol.
6 Spin the precipitated DNA again (12,000
rpm, 10 min, 4°C). Discard the
supernatant and lyophilize the pellet.
7 Add 6 µl deionized formamide to the
lyophilizate and resuspend the probe
DNA by vigorously shaking the tube
for > 30 min at room temperature.
8 Add 6 µl of hybridization buffer to the
tube containing probe DNA and again
shake for > 30 min.
9 Denature and dehydrate normal metaphase
chromosomal DNA on slides as
described in the literature (Lichter and
Cremer; Lichter et al., 1990).
10 Prepare the probe (from Step 8) for
hybridization by performing the
following steps:
Denature probe DNA at 75°C for
5 min.
Preanneal repetitive sequences by
incubating the probe at 37°C for
20–30 min.
11 Add prepared probe (from Step 10) to
denatured chromosomal spreads (from
Step 9). Proceed with in situ hybridization
and probe detection as described
in the literature (Lichter and Cremer;
Lichter et al., 1990).
12 Evaluate the CGH experiment with an
epifluorescence microscope.
Results
The CGH procedure was used to reveal
chromosomal imbalances (Figure 3) in a
T-cell prolymphocytic leukemia (T-PLL).
Chromosomal regions 6q, 8p, and 11qter
are stained more weakly in the tumor
DNA than in the control DNA, indicating
deletions of all or part of the chromosomal
arms in the tumor DNA. CGH also
identified overrepresented chromosomal
regions (6p, 8q, and 14q) which stain more
heavily in the tumor than in the control.
CONTENTS
INDEX