31 Multicolor FISH Techniques for the Detection of Inter- and Intrachromosomal Rearrangements
417
All probe DNAs are labeled by random priming using the BioPrime kit
with slight modifications to the protocol (Weier et aI1995a,b; Chapter 18,
Quantitative DNA Fiber Mapping by Heinz-Ulrich Weier).
Probes to be visualized in red are labeled by incorporation of digoxigenin-ll-dUTP (B.M.) at a digoxigenin-ll-dUTP:dTTP ratio of 35:65.
Therefore, I mM digoxigenin-ll-dUTP and 1 mM dTTP solution are simultaneously added in the random priming reaction.
Biotin-labeled probe DNA is prepared using a nucleotide mixture provided with the BioPrime kit, while fluorescein-12-dUTP (fluorescein-12dUTP:dTTP ratio of 35:65) is used in the random priming mixture for
probes to be visualized in green.
Biotinylated probes are detected with avidin-Cy5, which fluoresces in
the infrared spectrum when excited around 640 nm.
Yellow visualization is obtained using probes which have been random
primed in the presence of 1 mM digoxigenin-ll-dUTP and 1 mM fluorescein-12-dUTP at a ratio of 50:50.
The hybridization mixture contains 70 ~l of human COT-l DNA, 17 ~l
of salmon sperm DNA and between 60-120 ng of each probe, depending on
hybridization quality and signal intensity ofindividual probes. The DNA is
precipitated in ethanol and resuspended in 3 ~l water, before 7 ~l of Master Mix 2.1 (Weier et al1995b) is added so that the probe is dissolved in
55% formamide, 10% dextran sulfate and 2xSSC, pH 7.0. The probe mixture is then denatured by heating to 75°C for 7 min and placed at 37°C for
30 min. Slides are denatured at 75°C for 3.5 min in denaturing solution
and dehydrated in a 70%,85%,100% ethanol series. About 10 ~l of hybridization mixture are placed on top of the cells, covered with a 22 x 22 mm
coverslip, sealed with rubber cement and incubated for 44-48 h at 37°C in a
humidified chamber.
Posthybridization washes are performed as described previously
(Weier et al., 1995a, 1995b), including three washes in PN buffer. Unspecific binding sites are blocked with PNM solution (Pinkel et al., 1986). The
different layers of fluorochrome labeled avidin or antibodies (AB) are applied in the concentrations listed in Table 3. The yellow color is generated
by overlapping green and red signals, i.e., by hybridization of probes
labeled simultaneously with digoxigenin and FITe. After each AB layer,
slides undergo three 10-min washes in PN buffer with agitation and are
again blocked with PNM. After the final wash in PN buffer, slides are
washed 10 min in 2xSSC/0.l% Tween 20 buffer, mounted with DAPI
(0.1 ~g/ml) in antifading solution and coverslipped.
417
All probe DNAs are labeled by random priming using the BioPrime kit
with slight modifications to the protocol (Weier et aI1995a,b; Chapter 18,
Quantitative DNA Fiber Mapping by Heinz-Ulrich Weier).
Probes to be visualized in red are labeled by incorporation of digoxigenin-ll-dUTP (B.M.) at a digoxigenin-ll-dUTP:dTTP ratio of 35:65.
Therefore, I mM digoxigenin-ll-dUTP and 1 mM dTTP solution are simultaneously added in the random priming reaction.
Biotin-labeled probe DNA is prepared using a nucleotide mixture provided with the BioPrime kit, while fluorescein-12-dUTP (fluorescein-12dUTP:dTTP ratio of 35:65) is used in the random priming mixture for
probes to be visualized in green.
Biotinylated probes are detected with avidin-Cy5, which fluoresces in
the infrared spectrum when excited around 640 nm.
Yellow visualization is obtained using probes which have been random
primed in the presence of 1 mM digoxigenin-ll-dUTP and 1 mM fluorescein-12-dUTP at a ratio of 50:50.
The hybridization mixture contains 70 ~l of human COT-l DNA, 17 ~l
of salmon sperm DNA and between 60-120 ng of each probe, depending on
hybridization quality and signal intensity ofindividual probes. The DNA is
precipitated in ethanol and resuspended in 3 ~l water, before 7 ~l of Master Mix 2.1 (Weier et al1995b) is added so that the probe is dissolved in
55% formamide, 10% dextran sulfate and 2xSSC, pH 7.0. The probe mixture is then denatured by heating to 75°C for 7 min and placed at 37°C for
30 min. Slides are denatured at 75°C for 3.5 min in denaturing solution
and dehydrated in a 70%,85%,100% ethanol series. About 10 ~l of hybridization mixture are placed on top of the cells, covered with a 22 x 22 mm
coverslip, sealed with rubber cement and incubated for 44-48 h at 37°C in a
humidified chamber.
Posthybridization washes are performed as described previously
(Weier et al., 1995a, 1995b), including three washes in PN buffer. Unspecific binding sites are blocked with PNM solution (Pinkel et al., 1986). The
different layers of fluorochrome labeled avidin or antibodies (AB) are applied in the concentrations listed in Table 3. The yellow color is generated
by overlapping green and red signals, i.e., by hybridization of probes
labeled simultaneously with digoxigenin and FITe. After each AB layer,
slides undergo three 10-min washes in PN buffer with agitation and are
again blocked with PNM. After the final wash in PN buffer, slides are
washed 10 min in 2xSSC/0.l% Tween 20 buffer, mounted with DAPI
(0.1 ~g/ml) in antifading solution and coverslipped.