Neuroblastoma Breakpoint Figure 1. Cloning of the translocation breakpoints of the constitutional t(1;17) translocation. (A) Schematic overview of the sequence of a cosmid contig spanning the breakpoint [18]. Nine repeat-free probes (white boxes) were used in Southern hybridizations. Recognition sites for PvuII (P) and EcoRI (E) are shown. (B) Southern blot analysis of genomic DNA of human-hamster cell hybrids containing either the der(1) (hybrid 32-7A) or the der(17) chromosome (hybrid 32-2F53VIII). Left panel: hybridization of probe 6 to PvuII-digested genomic DNA yielded a band of ,4 kbp in the 32- 2F53VIII cell line (arrow) but not in the control samples. Right panel: hybridization of probe 9 to EcoRI-digested genomic DNA showed an additional band of ,17 kbp in the 32-7A cell line (arrow) but not in the control sample. A/3: parental hamster cell line; normal: normal human genomic placenta DNA. (C) Schematic overview of a fragment of derivative chromosome 17. Two primers located in probe 6 (GSP1 and GSP2) were used in combination with adaptor primers to amplify breakpoint-spanning fragments from genomic DNA digested with BamHI (B), DraI (D), EcoRV (EV), PvuII (P) or StuI (S). All the resulting fragments contained sequences derived from chromosome 1, with an overlap of 3,134 bp. Seven nucleotides of unknown origin are inserted at the breakpoint site. The location of the t(1;17) translocation breakpoint is indicated by a vertical double-pointed arrow. White boxes represent chromosome 17 sequences, and black boxes represent chromosome 1 sequences. (D) Schematic overview of a fragment of derivative chromosome 1. Two primers located in or nearby probe 9 (GSP3 and GSP4, respectively) were used in combination with adaptor primers to amplify breakpoint-spanning fragments from genomic DNA digested with DraI (D), EcoRV (EV), PvuII (P) or StuI (S). All the resultant fragments contained sequences derived from chromosome 1, with an overlap of 3,845 bp. Four nucleotides of unknown origin are inserted at the breakpoint site. The translocation breakpoint and sequences are indicated as in panel (C). doi:10.1371/journal.pone.0002207.g001 Primer pair GSP4 and AP2 amplified products of 1,268 bp and 4,355 bp from, respectively, the 32-7A/SspI and 32-7A/PvuII GenomeWalker libraries. All four 32-7A-specific products were cloned, and complete sequencing showed overlapping novel sequences derived from chromosome 1 in all clones. In total, we sequenced 4,512 nucleotides, of which 3,845 bp were derived from chromosome 1 (GenBank Acc Nu AF379606; partly shown in Figure 2B). PLoS ONE | www.plosone.org 4 May 2008 | Volume 3 | Issue 5 | e2207