BMC Genomics 2004, 5:65 http://www.biomedcentral.com/1471-2164/5/65 clone was prepared from 2.5 ml cultures using a BAC Real- Prep (Qiagen, Valencia, CA) protocol. Two hundred nan- ograms from each sample were labeled using nick translation to incorporate one of five fluorochromes, Spectrum Red/Orange/Green dUTP (Vysis, Downers Grove, IL), diethylaminomethylcoumarin (DEAC)-5- dUTP (NEN/Perkin Elmer Life Sciences, Boston, MA), or Cy5-dUTP (Amersham Biosciences, Piscataway, NJ). Typ- ically, 25 ng of each of five differentially labeled probes were pooled and precipitated in the presence of 15 µg of sonicated genomic dog DNA as competitor. Chromosome preparation, probe hybridization and post hybridization washes were performed as described previously [35,36]. Chromosomes were counterstained in 80 ng/ml 4', 6-dia- midino-2-phenylindole (DAPI) and mounted in anti-fade solution (Vectashield, Vector Laboratories, Burlingame, CA). Images were acquired and processed using a multi- color FISH workstation comprising a fluorescence micro- scope (Axioplan 2ie, Zeiss) equipped with narrow pass fil- ter sets and a cooled CCD camera (CoolSnapHQ, Photometrics, Tuscon, AZ) both driven by dedicated soft- ware (SmartCapture 2.3.1 Digital Scientific, Cambridge, U.K.). The digital image of each DAPI stained metaphase spread was processed using a high-pass spatial filter to reveal enhanced DAPI bands. Clones were assigned to a chromosome region according to the DAPI banded nomenclature of Breen et al. [35,36]. Refinement of probe order along the length of each chromosome was made by subsequent rehybridization to elongated canine chromosome preparations and/or by reference to inter- phase FISH analysis. Additional information may be found at http://www.cvm.ncsu.edu/mbs/ breen_matthew.htm. Alignment of dog BAC clones to orthologous regions of the human genome Nineteen 384-well plates of BAC clones from the RPCI-81 library [27] were selected at random, and end-sequence data were obtained from each clone using previously described methods [1]. Paired end-sequences for 1910 clones were masked for repetitive elements and searched against the human genome (NCBI build 31, November 2002, http://www.ncbi.nlm.nih.gov/). For 648 of the BACs (34%), each of the paired end-sequences gave a best hit to human genomic locations that are separated by 50– 500 Kb, and aligned head-to-head (mean span of human genomic DNA = 191 Kb). The remaining 1262 BAC-end sequences were searched against scaffolds of the 1.5x assembly [2] using wu-blastn (matrix = identity, W = 40) to identify scaffold sequences that contained at least short overlaps (40 bases) of identical sequence. For 954 of the 1262, hits were detected for both of the paired end- sequences. The homologous scaffold sequences were trimmed to remove any sequence that extended beyond 5 Kb from the region of alignment. They were then masked for repetitive elements, and searched against the human genome using wu-blastn (E<0.1). Again, only the best hit was considered. For 604 of the BACs (32% of the original sample), the paired scaffolds gave the best hits to genomic locations that are separated by 50–500 Kb (mean = 202 Kb), and aligned with their component BAC-end sequences in a head-to-head orientation. Altogether a total of 1252 (648+ 604) canine paired BAC-end sequences demonstrated significant hits with the human sequence. Accession numbers, PCR conditions, primers and BAC- end sequences are available for all markers at: http:// www-recomgen.univ-rennes1.fr/doggy.html and http:// www.fhcrc.org/science/dog_genome/dog.html. Abbreviations BAC-Bacterial Artificial Chromosome FISH-Fluorescence in situ Hybridization PCR-Polymerase Chain Reaction RH-Radiation Hybrid CGH-Comparative Genomic Hybridization cR-centiRays Mb-megabases Kb-kilobases Bp-base pair Min.-minutes E. Coli-Escherichia coli SOM-Supporting Online Material DAPI-Diamidino-2-phenylindole CS-Conserved Segments CSO-Conserved and Ordered Segments TIGR-The Institute for Genomics Research FHCRC-Fred Hutchinson Cancer Research Center Authors' contributions MB, RT, AS, and RH all contributed to the FISH mapping aspects of the manuscript, including isolation of BAC DNA samples, generation of canine chromosome Page 9 of 11 (page number not for citation purposes)