In Situ Hybridization Protocols Second Edition Edited by Ian A. Darby Methods in Molecular Biology Methods in Molecular Biology TM TM VOLUME 123 HUMANA PRESS HUMANA PRESS In Situ Hybridization Protocols Second Edition Edited by Ian A. Darby Human Partial Chromosome Paints 3 3 From: Methods in Molecular Biology , Vol. 123: In Situ Hybridization Protocols Edited by: I. A. Darby © Humana Press Inc., Totowa, NJ 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 4 Archidiacono et al. 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- 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. Human Partial Chromosome Paints 5 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. 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 (A and C) and Cy3 signals (B and D) 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. 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 mM each, pH 7.0; b. 10× Reaction buffer (usually comes with the Taq polymerase); c. Taq polymerase (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/µL each). 9. TBE buffer (900 mM Tris-base, 900 mM boric acid, 1 mM EDTA). 2.2 Nick-Translation 1. 10× Buffer: 0.5 M Tris-HCl, pH 7.8–8.0, 50 mM MgCl 2 , 0.5 mg/mL bovine se- rum albumin (BSA). 2. dNTPs mix: 0.5 mM dATP, 0.5 mM dCTP, and 0.5 mM dGTP. 3a. Biotin-11-dUTP mix: 0.5 mM dTTP and 0.5 mM bio-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) (see Note 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 mM sodium chloride, 15 mM sodium 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 µL of H 2 O, 200 µL of 1 M Tris-HCl, 9 mL of glycerol. 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 µL of DNA from hybrid; b. 37.2 µL of H 2 O; c. 5 µL of PCR buffer 10×; d. 5 µL of 10× dNTPs mix; e. 0.8 µLofTaq polymerase (5 U/µL) (AmpliTaq Gold, Perkin-Elmer); and f. 0.5 µL for each primer (1 µM final 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 (see Note 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-µL aliquot on 1% agarose gel. Store at 4°C (see Fig. 3). 3.2. Probe Labeling by nick-translation 1. Add to a microfuge tube, on ice: a. 2 µg of amplified products; b. 10 µL of 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) (see Note 1); 8 Archidiacono et al. e. 10 µL of 0.1 M β-mercaptoethanol; f. Dilute (immediately before use) 1 µL of 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; see step 4); g. 1.5 µL of 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-µL aliquot of each sample; add 4 µL of H 2 O and 1 µL of 10× gel loading buffer. Run on 1% agarose gel to check fragment size (see Note 3). 5. Stop the reaction by adding 4 µL 0.5 M EDTA. The labeled probe can be stored frozen for several months. 3.3. Probe Denaturation 1. Precipitate 20 µL of labeled DNA (400 ng) with 5 µg of human Cot-I DNA (see Note 4), 3 µg of salmon sperm DNA, 0.8 µL of 3 M Na 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 µL per slide). To a test tube add: 5 µL of deionized formamide, 2 µL of 50% dextran sulfate, 2 µL of distilled water, and 1 µL of 20× SSC. If more slides have to be hybridized, a master mix can be prepared. Fig. 3. Samples of Alu-PCR amplification products from four different hybrids. The PCR reaction was done in a volume of 50 µL. 5 µL were 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. Human Partial Chromosome Paints 9 3. Resuspend pellet in 10 µL of 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 µL of 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 µL of 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 µL of 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 5 are 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) (see Note 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. (See Note 6 and Figs. 2 and 3 for 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. [...]... Various combinations of protein markers on the chromosome and DNA probes have been used to investigate the meiotic chromosomal pairing process; the role of core protein in homologous chromatin domain interactions; as well as the relationship between chromosomal pairing, synapsis, and segFrom: Methods in Molecular Biology, Vol 123: In Situ Hybridization Protocols Edited by: I A Darby © Humana Press Inc.,... Radioactive In Situ Hybridization to Animal Chromosomes Graham C Webb 1 Introduction Although largely replaced by the use of fluorescent in situ hybridization (FISH) in animal and human molecular cytogenetics, the technique of radioactive in situ hybridization (RISH) still has some uses Using practicable exposure times for autoradiographs of 3–4 wk, RISH is approx 50 times more sensitive than FISH using biotin-... h mS-mit interval 4 Add colchicine solution (see Subheading 3.1.2.), 10–20 min if step 2 is taken; otherwise 30–60 min 5 Harvest in the common manner (see Subheading 3.1.2.) 3.1.4 Incorporation of 5-BrdU into Cultured Bovine Lymphocytes Cattle, sheep, and deer seem to be sensitive to high levels of 5-BrdU or thymidine 1 To produce mainly GBG-bands follow Subheading 3.1.2 but use an arresting dose of... and air dry Continue with the in situ procedures 3.3 FISH Detection 3.3.1 Probe Labeling Detailed protocols for probe purification can be found in Heng and Tsui (5) 3.3.1.1 BIOTIN LABELING 1 Purified DNA (1 µg) is labeled with a BRL BioNick kit according to the supplier’s instructions (15°C for 60 min for probes of 1–40 kb; 120 min for probe >100 kb) Optional digoxigenin (DIG) labeling of DNA (1 µg)... solution C 3× for 3 min each in a fresh solution 6 Apply 30 µL of blocking solution per slide Cover with a plastic coverslip and incubate at room temperature for 5 min 7 Apply 30 µL of biotinylated goat anti-avidin antibody working solution per slide and cover with a plastic coverslip Incubate at 37°C for 20 min 8 Following removal of the coverslip, wash the slides 3× for 3 min each in wash solution C... TIG 7, 149–154 4 Spyropoulos, B and Moens, P B (1994) In situ hybridization of meiotic prophase chromosomes, in Methods in Molecular Biology: In Situ Hybridization Protocols (Choo, K H., ed.), Humana Press, Totowa, NJ, pp 131–139 5 Heng, H H Q and Tsui, L.-C (1994) FISH detection on DAPI banded chromosomes, in Methods in Molecular Biology: In Situ Hybridization Protocols (Choo, K H., ed.), Humana Press,... and interphase cells by in situ suppression hybridization using recombinant DNA libraries Hum Genet 80, 224–234 3 Collins, C., Lin Kuo, W., Segraves, R., Fuscoe, J., Pinkel, D., and Gray, J W (1991) Construction and characterization of plasmid libraries enriched in sequences from single human chromosomes Genomics 11, 997–1006 Human Partial Chromosome Paints 13 4 Vooijs, M., Yu, L.-C., Tkachuk, D., Pinkel,... Parafilm-lined floor of a humid chamber 20 Heng, Spyropoulos, and Moens 3 Place the slide in a small humid chamber located inside a plastic bag containing a PBSsoaked tissue Incubate in the dark at room temperature overnight or at 37°C for 1 h 4 Repeat the washing procedure described in step 1 Add the secondary antibody as described in step 2 and incubate at 37°C for 1 h 5 Wash 3 × 10 min each in PBS... long Such short probes should be excised from cloning vectors If the probe sequence is left in the vector, groups of silver grains may be present in the final autoradiograph instead of single grains, as both signal and background noise, resulting in a very messy slide which is usually difficult or impossible to interpret This clustering of silver grains seems to be related to electrostatic charge and... minimal light exposure.) 5 Remove coverslips and wash the slides in wash solution C 3× for 3 min each 6 Stain with DAPI by immersing the slides in 0.2 µg/mL of DAPI in 2× SSC for 5– 10 min at room temperature Rinse in 2× SSC 3× for 2 min each 7 Mount the slides with 20 µL of antifade solution Cover with a 22 × 40 mm glass coverslip Apply gentle downward pressure to flatten the coverslip before examining . Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppres- sion hybridization using recombinant DNA libraries. Hum. Genet. 80, 224–234. 3. Collins, C., Lin. combinations of protein markers on the chromosome and DNA probes have been used to investigate the meiotic chromosomal pairing pro- cess; the role of core protein in homologous chromatin domain interactions; as. 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