In Situ Hybridization Protocols

334 Pages·2000·3.968 MB·English

by  Ian A. Darby (ed.)

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Human Partial Chromosome Paints 3
1
Preparation of Human Partial Chromosome Paints
from Somatic Cell Hybrids
Nicoletta Archidiacono, Rosalia Marzella, Cosma Spalluto,
Margherita Pennacchia, Luigi Viggiano, and Mariano Rocchi
1. Introduction
Whole chromosome painting libraries (WCPLs) have provided a very pow-
erful tool to cytogeneticists. The technique allows the painting of specific chro-
mosomes in metaphase spreads and in interphase nuclei (1–4). The usefulness
of WCPLs is particularly evident in identifying the chromosomal origin of de
novo unbalanced translocations and marker chromosomes, or, more generally,
in characterizing those cytogenetic cases in which the conventional approach
based on banding techniques failed to elucidate the chromosomal rearrange-
ment under study (5,6). Such cases are frequently experienced in cancer cyto-
genetics(7,8). WCPLs are usually derived from flow-sorted chromosomes.
Biotinylated genomic DNA from hybrid cell lines retaining specific human
chromosomes has been alternatively used as starting material to obtain WCPL
(9,10). Human chromosomes, however, represent only a minor component of a
human–rodent somatic cell hybrid, so that the sensitivity of this technique is
usually unsatisfactory (10). These problems can be circumvented by selective
polymerase chain reaction (PCR) amplification of the human sequences using
accurately designed dual-Alu primers (11,12). The amplified PCR products are
then labeled and used in fluorescent in situ hybridization (FISH) experiments
(reverse-FISH).
Human–rodent somatic cell hybrids frequently retain fragments of human
chromosomes as a consequence of rearrangements that occurred in vitro. This is
particularly true for human–hamster somatic cell hybrids, because hamster chro-
mosomes are prone to rearrangements. We have recently characterized, by
reverse-FISH, hundreds of human-hamster hybrids, and have identified a large
From:Methods in Molecular Biology, Vol. 123: In Situ Hybridization Protocols
Edited by: I. A. Darby © Humana Press Inc., Totowa, NJ
3
4 Archidiacono et al.
Fig. 1. Summary of our collection of fragments specific for chromosome 12. Each
fragment is represented by a solid bar specifying its length and subchromosomal loca-
tion on a G-band ideogram of chromosome 12. The code number for each hybrid is
also shown. The hybrids are grouped in two categories: (1) 12HY: classical
(nonradiation) hybrids. The rearrangement occurred in vitro by chance. Whole chro-
mosomes or additional fragments from different chromosomes are present in these
hybrids. (2) 12RH: radiation hybrids derived from the monochromosomal hybrid
Y.E210TC (generated in our laboratory), retaining chromosome 12 as the only human
contribution.
number of hybrids containing chromosome fragments. These hybrids can be
used as starting material for the production of partial chromosome paints (PCPs),
that is, paints that recognize a specific chromosomal region. They can be used
as a powerful tool for cytogenetic investigations (13,14). Most of these hybrids,
however, contain additional fragments and/or entire human chromosomes, which
prevents their efficient use. It would be therefore advantageous to obtain hy-
brids retaining a single fragment as the only human contribution. To generate
these type of PCPs, we have started to produce radiation hybrids (RHs) from
monochromosomal hybrids (15). The diagram shown in Fig. 1 summarizes as
an example the PCPs from normal hybrids and RHs, specific for chromosome
12. To date RHs specific for chromosomes 2, 4, 5, 7, 10, 12, 17, 19, and X have
been obtained. Thanks to support from AIRC and Telethon, DNA from these
hybrids is available to the scientific community free of charge. FISH images of
these PCPs can be viewed at the Internet site http://bioserver.uniba.it/fish/Cyto-
Human Partial Chromosome Paints 5
Fig. 2. FISH images of hybrids no. 425 (A–B), specific for 12p11.2-pter and no.
424(C–D)specific for 12q. The DAPI banding (AandC) and Cy3 signals (BandD)
are reported separately, in black and white, as captured by the CCD camera. DAPI-
banded chromosome 12 and the corresponding Cy3 signals are shown side by side in
the two boxes.
genetics/welcome.html). Figure 2 shows, as examples, PCPs no. 425 specific
for 12p11.2→12pter, and no. 424 specific for 12q.
Alu and long intervening elements (LINE) sequences are not evenly
interspersed in the human genome; their primary distribution correlates with
G-negative and G-positive bands respectively (16), so that Alu-PCR products
generate a banding pattern corresponding to R-banding (17). This fact has to
be kept in mind in evaluating the FISH signals of PCP obtained with the
present approach.
6 Archidiacono et al.
2. Materials
2.1. PCR
1. Alu primers (11):
5' GGATT ACAGG YRTGA GCCA 3' (Y = C/T; R = A/G)
5' RCCAY TGCAC TCCAG CCTG 3'
stored frozen as 100 pmol/µL.
2. Genomic DNA from hybrid (100 ng) in distilled water, stored frozen.
3. 0.5-mL Test tubes suitable for the thermal cycler.
4. A set of micropipets (P20 and P200, Gilson, Villiers-le-Bel, France) and
sterile tips.
5. Reagents for PCR (stored frozen):
a. 10× dNTPs mix 2 mMeach, pH 7.0;
b. 10×Reaction buffer (usually comes with the Taqpolymerase);
c. Taqpolymerase (5 U/µL) (AmpliTaq Gold, Perkin-Elmer);
d. Sterile distilled water (autoclaved), stored at room temperature; and
e. Light mineral oil (not necessary if the thermal cycler is equipped with a
heated lid).
6. Programmable thermal cycler.
7. Agarose; ethidium bromide; gel electrophoresis apparatus, and UV transillumi-
nator.
8. Molecular weight marker (λ-HindIII + PhiX–HaeIII, 50 ng/µLeach).
9. TBE buffer (900 mMTris-base, 900 mMboric acid, 1 mMEDTA).
2.2 Nick-Translation
1. 10×Buffer: 0.5 MTris-HCl, pH 7.8–8.0, 50 mMMgCl , 0.5 mg/mL bovine se-
2
rum albumin (BSA).
2. dNTPs mix: 0.5 mMdATP, 0.5 mMdCTP, and 0.5 mMdGTP.
3a. Biotin-11-dUTP mix: 0.5 mMdTTP and 0.5 mMbio-11-dUTP (or bio-16-dUTP);
b. Alternatively: digoxigenin (DIG)-11-dUTP mix (Boehringer Mannheim) (see
Note 1);
c. Alternatively (for direct labeling): dUTP-Cy3 (Amersham) (seeNote 1).
4. Enzymes: DNA polymerase I (5 U/µL) and DNase I (2 U/µL).
5. Other chemicals: 0.1 M β-mercaptoethanol and 0.5 M EDTA.
2.3.In Situ Hybridization
1. Cot-I DNA, human (BRL or Boehringer Mannheim).
2. Salmon sperm DNA, 1 µg/µL.
3. 3 M Na acetate.
4. 70%, 90%, and 100% ethanol.
5. Savant concentration centrifuge.
6. Formamide and deionized formamide.
7. 50% Dextran sulfate, autoclaved.
8. 20× SSC (1× SSC = 150 mMsodium chloride, 15 mMsodium citrate, pH 7.0).
Human Partial Chromosome Paints 7
9. Vortex.
10. Coplin jar.
11. 24 × 24 mm and 24 × 50 mm coverslips.
12. Rubber cement.
13. Washing solution A: 50% formamide/2× SSC.
14. Washing solution B: 0.1× SSC.
15. Blocking solution: 3% BSA, 4× SSC, 0.1% Tween-20.
16. Detection buffer: 1% BSA, 1×SSC, 0.1% Tween-20.
17. 1 mg/mL of Avidin-Cy3 (Amersham) (or 1 mg/mL fluorescein isothiocyanate
[FITC]-conjugated anti-DIG antibodies [Boheringer Mannheim]).
18. Solution C: 4× SSC, 0.1% Tween-20.
19. DAPI (4',6-diamidino-2-phenylindole, Sigma).
20. Propidium iodide (PI) (Sigma).
21. Antifade-mounting medium. 10 mL: 0.233 g of DABCO (1,4-diazabicyclo-
[2.2.2]octane, Sigma), 800 µLof H O, 200 µLof 1MTris-HCl, 9 mL of glycerol.
2
3. Methods
3.1. Generation of PCR Products from Somatic Cell Hybrids
1. In a 0.5-mL tube (suitable for a thermocycling machine) mix in this order:
a. 1 µLof DNA from hybrid;
b. 37.2µL of H O;
2
c. 5 µLof PCR buffer 10×;
d. 5 µL of 10×dNTPs mix;
e. 0.8 µLofTaqpolymerase (5 U/µL) (AmpliTaq Gold, Perkin-Elmer); and
f. 0.5 µLfor each primer (1 µMfinal concentration).
g. Adjust total volume to 50 µL.
h. Overlay with a drop of light mineral oil (this step is not necessary if the ther-
mal cycler is equipped with a heated lid).
2. In a second tube: add everything as described above, omitting the DNA. This
serves as a negative control. (If more than one sample is amplified, a master mix
can be prepared.)
3. Run on the thermocycling machine as follows:
a. 10 min at 95°C (seeNote 2);
b. 30 cycles: 1 min at 94°C, 1 min at 65°C, 4 min at 72°C; and
c. 10 min at 72°C.
4. Check the amplification products by loading a 5-µLaliquot on 1% agarose gel.
Store at 4°C (seeFig. 3).
3.2. Probe Labeling by nick-translation
1. Add to a microfuge tube, on ice:
a. 2 µg of amplified products;
b. 10µLof 10×nick-translation buffer;
c. 10 µL of dNTPs mix;
d. 5 µL of biotin mix (or 5 µL DIG mix; or 1 µL of dUTP-Cy3 mix) (seeNote 1);
8 Archidiacono et al.
Fig. 3. Samples of Alu-PCR amplification products from four different hybrids.
The PCR reaction was done in a volume of 50 µL. 5 µLwere then run on a 1% agarose
gel, at 100 V. The marker in lane 6 is λ-HindIII, the one on lane 5 is pCMVβHaeIII
(18). The first five bands of the latter are: 970, 750, 595, 544, and 447 bp. Amplified
fragments range approximately from 3–4 kb to 300 bp. Discrete bands are sometimes
detected in amplified DNA from hybrids which retained a small amount of human
material. The run is also informative for a quantitative evaluation of PCR products,
calculated approximately by visual comparison with the known amount of the marker.
Quantitative evaluation can be performed better by inspecting the gel after a few min-
utes of running, when both the marker and the samples appear as compact bands.
e. 10 µL of 0.1Mβ-mercaptoethanol;
f. Dilute (immediately before use) 1 µLof DNase I in 1mL of distilled water;
add 20 µL to the nick-translation mix. (The DNase I should be calibrated to
give fragments of 100–500 bp; seestep 4);
g. 1.5 µLof DNA polymerase I; and
h. Sterile distilled water to 100 µL.
2. Incubate at 15°C for 2 h.
3. Place at 4°C until checked on gel.
4. Take a 5-µLaliquot of each sample; add 4 µLof H O and 1 µLof 10×gel loading
2
buffer. Run on 1% agarose gel to check fragment size (seeNote 3).
5. Stop the reaction by adding 4 µL0.5MEDTA. The labeled probe can be stored
frozen for several months.
3.3. Probe Denaturation
1. Precipitate 20 µLof labeled DNA (400 ng) with 5 µg of human Cot-I DNA (see
Note 4), 3 µg of salmon sperm DNA, 0.8 µLof 3MNa acetate, and 3 vol of cold
(–20°C) ethanol. Leave at –80°C for 15 min. Centrifuge for 15 s (12,000g) at
4°C. Dry the pellet on a Savant centrifuge for a few minutes.
2. Prepare hybridization mix (10 µLper slide). To a test tube add: 5 µLof deionized
formamide, 2 µLof 50% dextran sulfate, 2 µLof distilled water, and 1 µLof 20×
SSC. If more slides have to be hybridized, a master mix can be prepared.
Human Partial Chromosome Paints 9
3. Resuspend pellet in 10 µLof hybridization mix, by vortexing.
4. Denature DNA mix at 80°C for 8 min. Transfer to 37°C for 20 min, then place on
ice until used.
3.4. Slide Denaturation
1a. Prepare 50 mL of denaturing solution (70% deionized formamide/2×SSC). Pour
into a Coplin jar. Place in a water bath at 70°C. Check the temperature inside the
jar.
2a. Prewarm slides at 60°C in dry heat oven.
3a. Immerse the slides in the denaturation solution for exactly 2 min, two slides at a
time.
Alternative (faster) slide denaturation:
1b. Prepare 200 µL/slide of denaturing solution (70% deionized formamide/2×SSC).
2b. Prewarm slides at 60°C in dry oven.
3b. Put 200 µL of denaturation solution on each slide, cover with a 24 × 50 mm
coverslip, incubate for exactly 2 min at 80°C in a dry oven or on an appropriate
thermoblock plate.
4. Dehydrate slides in 70%, 90%, and 100% ethanol, 3 min in each solution (70%
ethanol at –20°C).
5. Dry slides after dehydration.
3.5. Hybridization
1. Apply 10 µLof hybridization mix to denatured slides, avoiding air bubbles.
2. Cover with 24 ×24 mm clean coverslip; seal with rubber cement.
3. Incubate in a moist chamber overnight at 37°C.
3.6. Posthybridization Washing and Detection
Do not allow slides to dry out at any stage. All washing is performed in a
Coplin jar.
1. Remove coverslips and wash 3×for 5 min in prewarmed solution A in a Coplin
jar in a shaking water bath at 42°C. This first step can be omitted without signifi-
cant difference.
2. Wash 3× for 5 min in prewarmed solution B in a water bath at 60°C.
3. Apply 200 µLof blocking solution per slide; cover with 24 ×50 mm coverslip;
transfer the slides in a moist chamber; incubate for 30 min at 37°C.
4. Dilute stock solution of avidin-Cy3 (1 mg/mL) 1:300 in detection buffer. If DIG
labeling has been performed: dilute stock solution of FITC-conjugated anti-DIG
(Boheringer Mannheim), according to the manufacturer’s instruction. Let cover-
slips slide off, then apply 200 µLof detection solution per slide. Cover with 24 ×
50 mm coverslips. Transfer the slides in a dark moist chamber. Incubate at 37°C
for 30 min. Important: the detection steps 3,4, and 5are not necessary if labeling
has been performed with dUTP-Cy3.
10 Archidiacono et al.
Fig. 4. (See Color Plate 1 following p. 176.) (A) Results of FISH experiments in a
case of pericentric inversion of chromosome 2. PCP no. 113 (specific for the region
2q11–2q23, red signal) and PCP no. 114 (specific for the short arm of chromosome 2,
green signal) have been used. Two pairs of homologs from distinct metaphases have
been shown. Normal chromosome 2 (N) is on the left. In the upper row the DAPI
banded chromosomes are shown without signal, to better illustrate their morphology.
continued next page
Human Partial Chromosome Paints 11
5. Remove the coverslips; rinse the slides 3×for 5 min each in prewarmed washing
solution C in a water bath at 42°C.
6. Counterstain with DAPI (200 ng/mL in 2×SSC) (seeNote 5) or with propidium
iodide (200 ng/mL in 2× SSC), or both.
7. Rinse for 2 min in 2× SSC 0.05% Tween-20 at room temperature.
8. Apply two drops of antifade-mounting medium and cover with 24 ×50 mm cov-
erslip. Slides can be stored for weeks or months in the dark at 4°C.
3.7. Fluorescence Microscopy
Signals from painted chromosomes are visible using an epifluorescence micro-
scope equipped with specific filters for the fluorochromes utilized. A 100 W or
preferably 50 W mercury high-pressure lamp is suitable. If a normal photographic
system is used, dual- or three-band pass filters are appropriate. Multiple-bandpass
filters, however, reduce the amount of light reaching the camera. If a black and
white cooled charged-couple device (CCD) camera is used, filters should be dis-
tinct for each fluorochome and aligned, to guarantee an exact merging of images.
Recent microscopes (such as Leica) mount perfectly aligned filters. If an older
microscope is used, the image shifting problem can be circumvented using a triple-
bandpass filter, a triple-band dichroic mirror, and distinct excitation filters (Chroma
Technology). Cooled CCD cameras are currently the most sensitive devices. Sig-
nals from painting probes, however, are usually strong enough to be recorded using
a conventional photographic camera. (SeeNote 6andFigs. 2and3for examples of
the applications of the present technique.)
4. Notes
1. Probe labeling can be performed using biotin-16-dUTP (Boehringer Mannheim),
Cy3-conjugated 11-dUTP, or digoxigenin-11-dUTP (Boehringer Mannheim). The
DIG-11-dUTP is available premixed in the appropriate ratio with dNTP. The 5 µL
The breakpoints were identified as 2p13 and 2q34. (B) These two examples show
FISH experiments performed to characterize the evolution of the phylogenetic chro-
mosome XVII. In the upper part, FISH experiments using PCPs nos. 2 and 35, specific
for the short and long arm of human chromosome, 17 respectively, show that a
pericentric inversion has differentiated the human (HSA, Homo sapiens) and pygmy
chimpanzee (PPA, Pan paniscus). In the lower part, PCPs no. 2 (red signal) and no.
497 (the latter specific for the 17q22-qter region, yellow signal) have been used to
characterize the paracentric inversion that differentiated HSA and orangutan (PPY,
Pongo pygmaeus). The original color of PCP no. 497 is green. This PCP, however, is
part of PCP no. 2. The yellow color is generated by the merging of red and green
signals. These analyzes suggest that the breakpoints of the pericentric inversion that
differentiated HSA and PPA are located at 17p11.2 and 17q21, while those of the
paracentric inversion found in PPY are located at 17q11.2 and 17q23.