Fluorescence In Situ Hybridization using Single-Molecule DNA Fibers (Fiber-FISH)
The probes used in this study included four fosmid clones selected from the UCSC Genome Browser GRCh37/hg19 assembly and a 3,632-bp PCR product that is specific for the glycophorin E repeat (see below). Probes were made by whole-genome amplification with GenomePlex Whole Genome Amplification Kits (Sigma-Aldrich) as described previously.32 Briefly, the purified fosmid DNA and the PCR product were amplified and then labeled as follows: G248P86579F1 and glycophorin E repeat-specific PCR product were labeled with digoxigenin-11-dUTP, G248P8211G10 was labeled with biotin-16-dUTP, G248P85804F12 was labeled with DNP-11-dUTP, and G248P80757F7 was labeled with Cy5-dUTP. All labeled dUTPs were purchased from Jena Bioscience.
The preparation of single-molecule DNA fibers by molecular combing and fiber-FISH was as previously published,3, 33 with the exception of post-hybridization washes, which consisted of three 5-min washes in 2× SSC at 42°C, instead of two 20-min washes in 50% formamide/50% 2× SSC at room temperature.