Methods of chromosome preparation 1.

Equipment The equipment required for the metaphase spreading protocol includes: (1) a source of hot steam (waterbath at 75-80 C); (2) a heated metal plate, ideally with a temperature gradient across its surface. To achieve this, the metal plate/lid covers the waterbath only partially (Fig. 1a), and carries a temperature gradient between the hot end and a cold end. A 2-3 mm thick metal plate is ideal. We tested plates made of stainless steel (3 mm and 1.5 mm thick) and copper (1 mm thick). When the plate rests about 2 cm from the hot water surface, the part of the plate in contact with the hot steam shows roughly 70 C, whereas the other end of the plate (10-15 cm long) is at room temperature. If such a metal plate is not available, a minimum of two surfaces, one at 65 -75 C (a simple heat block) and one at room temperature are required; (3) good quality slides. Two brands of pre-cleaned slides, Gold Seal (Becton, Dickinson Co, Portsmouth, NH) and Superfrost (Erie Scientific, Portsmouth, NH) were commonly used. They did not require any extra cleaning steps or washes prior to use. Cell suspensions were dropped on slides taken directly out of their original boxes. Lower quality glass-slides can be passed through successive washes in acetone, HCl/ethanol and triple distilled water and their quality compared.

Fig. 1.a: Waterbath image showing metal plate position, allowing both the temperature gradient formation and access to the hot steam; Fig.1b: Chemical aging setting: a few layers of gauze are placed in a plastic box (for example, a box used to store slides ). Two-three stripes of tape are placed across the opening to hold the gauze in place when the box is turned upside-down. Prior to use, the gauze is soaked in ethanol. Two-three freshly prepared slides 1

are placed on the metal block of a thermocycler. 200 ul ethanol are pipetted on each slide, and covered with a coverslip. The plastic box is placed upside-down on the slides, so that the wet gauze comes in contact with the slides and covers them (the gauze will prevent ethanol evaporation during the subsequent heating step). The slides are heated according to the sequence described in the text. After heating, the plastic box/cover and the coverslips are removed and the slides are air-dried.

2. CHROMOSOME PREPARATION. Cell culture and fixation. Cell suspensions from peripheral blood, fibroblasts, bone marrows, lymphoblastoid cell lines and germ cell tumors were used to prepare metaphases for G-banding and FISH. Cell culture was performed according to standard protocols. For harvesting, the hypotonic buffer used was 0.075M KCl, at 37 C for 10-20 minutes (incubations were shorter for peripheral blood cultures, longer for tumors). Hypotonic treatment increases cell volume, and disrupts the cell membrane of the red blood cells (allowing their removal). After hypotonic treatment, usually in 15 ml centrifuge tubes, cells were pelleted by centrifugation 10min at 1000 rpm and are resuspended in 1-1.5 ml fixative (3:1 methanol : acetic acid), then transferred into 1.5 ml vials. All subsequent fixative washes were done in these small vials, with centrifugations performed in a tabletop microcentrifuge for 1-2 minutes at 1500 rpm. Chromosome spreading and G-banding. Cells in fixative were diluted at the appropriate density, empirically determined in any cytogenetic laboratory, to produce a reasonable number of well-spread metaphases in each microscopic field. With an automatic pipette, 25-35l of cell suspension were evenly distributed on several locations on the slide and the liquid was spread by gently moving the pipette tip parallel to the surface. As the fixative gradually evaporated, the surface of the slide became grainy (cells visible). At that moment, the slide was placed face down into the steam of the hot water bath (75 C or more) for 1-3 seconds, then dried by placing the slide on the metal plate (carrying a gradient of temperature across its surface, Fig. 1a). The degree of spreading is adjusted using the different temperatures of the heated plate, with higher temperatures increasing chromosome spreading. For difficult to spread cells, after the surface became grainy, the slide was passed briefly through the water vapors, then 4-6 droplets of 2

acetic acid were placed on the slide. After the acetic acid slowly spreaded and covered the surface, the slide was held 3-5 seconds in the steam of the waterbath, then quickly dried on the hottest area of the metal plate or the metal block (65 C). After overnight incubation at 65 C (aging), G-banding was performed according to a standard laboratory procedure.

3. FISH: CHEMICAL AGING and SLIDE DENATURING. Chemical aging. A freshly prepared slide was placed on the metal block of a thermocycler (a PCR machine, Fig. 1b). 150-200l ethanol were pipetted on the slide, covered with a coverslip and ethanolsoaked gauze placed on top of them to prevent ethanol evaporation. The block was programmed to increase its temperature to 94 C, keep it for 2-20 seconds, then cool towards room temperature. Depending on the machine, the heating and cooling speed was 1-2 C/second. Alternatively, the slide can be incubated 10-15 seconds each, in jars with ethanol at 50 C, 75 C, 94 C, 75 C and 50 C, followed by drying at room temperature. After aging, the slides were subjected to brief (30-60 seconds) pepsin pretreatment, using 0.005% pepsin in 0.01N HCl., followed by rinsing in PBS and ethanol series, then air-drying. Gradual denaturing. "Simultaneous" protocol: (Fig. 2j-k). 11-12ul labeled DNA probe in hybridization buffer were pipetted on the slide, covered with a 22x22mm coverslip and sealed with rubber cement. The slide was placed on the metal block of a thermocycler, which was programmed to gradually (within 90 seconds) heat the slide to 75 C, keep that temperature for 90-120 seconds, and gradually (90 seconds) cool to room temperature. "Separate" protocol (Fig. 2i, 2l).. Labeled DNA probe is denatured 5 minutes at 75 C in a waterbath. For slide denaturing, 150 l of 70%formamide/2xSSC are pipetted on the slide, covered with a coverslip and the assembly is gradually heated on the metal block of the thermocycler as described above. Coverslip is discarded and the slide is rinsed through ethanol series, 3 minutes each in 70% and 100% ethanol at room temperature, then air-dried. For laboratories not equipped with a thermocycler, an alternative separate denaturing protocol uses three jars with 70%FA/2xSSC solution at 45 C, 60 C and 75 C. The slide is dipped 5-10 seconds each in the 45 C and 60 C solutions, then incubated 90-120 seconds at 75 C and dipped 3

again 5-10 seconds each in the 60 C and 45 C solutions, followed by brief rinsing in 70%, 100% ethanol and air drying. Probe labeling, and detection. Probes (centromeric repeats, chromosome paint probes, cosmids) were either purchased or were prepared and labeled by nick translation or PCR labeling, using commercially available (Boehringer Mannheim, Indianapolis, IN) or custom-made FITC-, biotin- or digoxigenindUTP. After overnight hybridization in a moist chamber at 37 C, the slides were subjected to one layer of detection for biotin (avidin-Cy3) and digoxigenin (anti-digoxigenin-FITC). Slides were examined with a Leica Aristoplan or an Olympus Provis AX70 microscope and images taken using cooled-CCD cameras (Photometrics, Tucson, AZ) and commercial software (PSI, Inc or Vysis, Inc). Fig. 3a-3ee: illustrates the processes taking place during chromosome spreading, as examined at the microscope using a 10x objective. In 3a-3e series, the pellet was placed directly on the slide, whereas in the 3aa-3ee series the cell suspension was dropped from about 1m distance. The letters correspond to the arbitrary five steps described in the text. Microscopically and macroscopically there is no difference in chromosome spreading between the two techniques. The drying technique is much more important. 3f-3i. (compare also with fig. 4). Cells resuspended in 3:1 methanol:acetic acid were dropped on slides and dried as follows: 2f, no water vapors, slide dried on the cold end of the metal plate (room temperature); 2g, slide exposed to water vapors, then dried at room temperature; 2h, no water vapors, slide dried on the hot end of the metal plate; 2i, slide exposed to water vapors, dried on the hot end of the metal plate.

Fig. 2. a-c: Simultaneous denaturing using hybridization buffer containing 12-15% dextran sulfate and different brands of formamide. Slides were chemically aged 10 seconds at 94 C on a thermocyler block and DAPI stained. Use of dextran-sulfate during denaturing increases chromosome thickness and alters their structure. Formamide brand influences the process as well, with chromosomes being either excessively thick (a) or with a distorted, uneven surfaces (b). Among the three images, the best brand of formamide was in (c), with chromosomes not as thick as (a) and more evenly stained than (b). d: Another slide from the same lot as above was subjected to separate denaturing for 2 minutes in a Coplin jar, containing 70%FA/2xSSC at 75 C (same formamide as in Fig. 2c). The absence of dextran sulfate results in less distorted chromosomes. However, the brightly stained centromeres indicate over-denaturing of the chromosomes, yielding a pseudo C-banding aspect. Also compare with (g), where separate denaturing using the same formamide brand was done gradually, on the metal block of a thermocycler. e-f: 10 minute hybridization of a probe for the alpha satellite repeat of chromosome 1, on cells chemically aged for 2 minutes. Arrows indicate weaker hybridization signals in (e) (no pepsin treatment) than in (f) (pepsin treated). g-h: 20 minutes hybridization of a biotinylated commercial probe for chromosome 1 (Oncor), detected with avidin FITC (g). Slide was subjected to 10 seconds chemical aging and separate denaturing on the metal block of a thermocycler. The grayscale DAPI image of the metaphase shown in (g) was inverted using Adobe Photoshop (h). Chromosomes 1, 5 and 19 (carrying hybridization signals) are identifiable. i: 10 second chemical aging and separate slide denaturing on the metal block of a thermocycler followed by 5 hours hybridization using a commercial diagnosis probe (Oncor, Inc) for Prader-Willi syndrome. j-k: 10 seconds chemical aging and simultaneous denaturing of a CGH analysis on the metal block of a thermocycler followed by 18 hours hybridization. Normal DNA was digoxigenin-labeled and detected by antidigoxigenin-FITC antibody (j). Tumor DNA (testicular germ cell tumor) was biotin-labeled and detected with Avidin Cy3 (k). Arrows show the typical 12p amplification characteristic for this tumors. Although the hybridization was even and of good quality, note the thickness of the chromosomes, mostly due to the presence of the dextran sulfate in the hybridization buffer during the direct denaturing phase. l: 10 seconds chemical aging and separate slide denaturing on a metal block of a thermocycler followed by 18 hours hybridization of the FITC-labeled painting probes of an M-FISH analysis.

Fig. 4. Pellet resuspended in 1:1 methanol:acetic acid (column a ), 6:1 methanol:acetic acid (column b) and 3:1 ethanol:acetic acid (column c). In row #1, no water vapors, slides dried on the cold end of the metal plate (room temperature); #2, slides exposed to water vapors, then dried at room temperature; #3, no water vapors, slide dried on the hot end of the metal plate; #4, slides exposed to water vapors, dried on the hot end of the metal plate. The best spreading and cleanest chromosome preparations are achieved in #1 and #4.

Fig. 5: Identical G-banding staining conditions on metaphases spread in various ways. Staining was done on all slides by keeping them 30" in 0.1% trypsin and 2 minutes in 1.5 % (v/v) Giemsa. Column a: cell suspension in 1:1 methanol:acetic acid; Column b, cell suspension in 3:1 methanol acetic acid. Row 1: slides prepared using water vapors but dried at room temperature; row 2 : slides prepared using water vapors and dried on the hot metal plate; row 3: slides prepared using no water vapors and dried at room temperature. Although banding worked in all conditions, the quality of the bands was a little better when slides were dried at room temperature compared to a heated surface. Also very visible is the increase in contrast of G-banding between cell suspensions in 1:1 fixative (a1,a2,a3) and 3:1 fixative (b1,b2,b3).

Legend: Fig. 5a: illustrates the darker Giemsa staining of metaphases of cells kept in 6:1 fixative (compare with fig. 5). b: reduced trypsin treatment of cells (3 seconds) results in thin chromosomes, uniformly stained by Giemsa, with little banding. c1 and c2: Trypsin treatment of long chromosomes. Long chromosomes (cells were in usual 3:1 fixative) require increased trypsin treatment to yield a better banding pattern. Trypsin time was increased from 30 seconds (c1) to 50 seconds (c2). d1 and d2: Trypsin treatment of short chromosomes. The short chromosomes depicted, were found on the same slides as the longer chromosomes above them, and thus were subjected to the same test of trypsin treatment (30 seconds in d1 and 50 seconds in d2). Short chromosomes did not respond well to long trypsin treatment, they became more "puffy" and lost the sharp edges. Shorter than 30 seconds trypsin treatment would have been more desirable for these shorter chromosomes.

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