Chromosomes and nuclei were spread onto glass slides following a standard procedure. Cell cultures (5 × 105 cells/ml) were incubated in medium containing colcemid (50 ng/ml, 30 min; 37°C; Sigma Chemical Co. , Ltd.), washed once in HBSS, once in PBS, swollen (0.075 M KCl, 15 min; 37°C), fixed with methanol/acetic acid (3:1), dropped (three drops; 2.5 × 106 cells/ml) onto washed slides, and then air dried.
To prepare extended DNA fibers (Parra and Windle, 1993 (link)), 2 μl cells resuspended in PBS (106 cells/ml) were spotted onto cleaned glass slides and lysed with 5 μl of 0.5% SDS in 200 mM Tris-HCl, pH 7.4, 50 mM EDTA (10 min, 20°C). Slides were tilted (15° to horizontal), allowing a stream of DNA to run slowly down the slide, air dried, and then fixed in methanol/acetic acid (3:1). For most purposes, cells containing halogenated DNA were diluted 30- and 100-fold with untreated HeLa cells, before spreading. This simplifies the spreads, allows isolated labeled DNA fibers to be found with relative ease, and makes possible the identification of replicons from a single labeled cell (see Fig.4 ).
To estimate the extension of DNA fibers, spreads were prepared from HeLa cells infected with adenovirus serotype 2 and grown in medium supplemented with 100 μM BrdU 15–20 h after infection (Pombo et al., 1994 (link)). Abundant Br-labeled DNA molecules measured 13.9 ± 1.3 μm (mean ± SD; n = 50). As the viral genome is 36 kbp, the extension of these DNA fibers is 2.59 ± 0.24 kbp/μm.
To prepare extended DNA fibers (Parra and Windle, 1993 (link)), 2 μl cells resuspended in PBS (106 cells/ml) were spotted onto cleaned glass slides and lysed with 5 μl of 0.5% SDS in 200 mM Tris-HCl, pH 7.4, 50 mM EDTA (10 min, 20°C). Slides were tilted (15° to horizontal), allowing a stream of DNA to run slowly down the slide, air dried, and then fixed in methanol/acetic acid (3:1). For most purposes, cells containing halogenated DNA were diluted 30- and 100-fold with untreated HeLa cells, before spreading. This simplifies the spreads, allows isolated labeled DNA fibers to be found with relative ease, and makes possible the identification of replicons from a single labeled cell (see Fig.
To estimate the extension of DNA fibers, spreads were prepared from HeLa cells infected with adenovirus serotype 2 and grown in medium supplemented with 100 μM BrdU 15–20 h after infection (Pombo et al., 1994 (link)). Abundant Br-labeled DNA molecules measured 13.9 ± 1.3 μm (mean ± SD; n = 50). As the viral genome is 36 kbp, the extension of these DNA fibers is 2.59 ± 0.24 kbp/μm.
