For DNA FISH, 0.5–1 × 106 lymphoblastoid cells were seeded on Poly-prep slides (Sigma) overnight. They were then fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized using 0.5% Triton X for 10 min66. Fosmid clones and plasmid were prepared and labeled as previously described67. Cells were denatured for 30 min. For four-color FISH, each slide was hybridized with between 80 and 100 ng of biotin-, digoxigenin- and directly labeled probes, 18 µg of human Cot1 DNA (Invitrogen) and 5 µg salmon sperm DNA. Green496-dUTP (Enzo Life Sciences) was used for direct labeling of fosmid probes. Washes and detection were as previously described67. See Supplementary Table 3 for Fosmid probe details.

Slides were imaged using a Photometrics Coolsnap HQ2CCD camera and a Zeiss AxioImager A1 fluorescence microscope with a Plan Apochromat 100×1.4NA objective, a Nikon Intensilight Mercury based light source and either Chroma #89014ET (three-color) or #89000ET (four-color) single excitation and emission filters (Chroma Technology Corp.) with the excitation and emission filters installed in Prior motorized filter wheels. A piezo electrically driven objective mount (PIFOC model P-721, Physik Instrumente) was used to control movement in the z dimension. Step size for z stacks was set at 0.2 µm. Nikon Nis-Elements software was used to perform hardware control, image capture, and analysis. Images were deconvolved using a calculated point spread function with the constrained iterative algorithm of Volocity (PerkinElmer). The quantitation module of Volocity was used to calculate interprobe distances. To eliminate the possibility of measuring sister chromatids, only alleles with single probe signals were analyzed.

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