Confirmation of breakpoint junctions generated by the chromosomal abnormalities. (A) Confirmation of the breakpoints in J20 by PCR. Genomic DNA from L1 (lane 1), L2 (lane 2), S1 (lane 3), S2 (lane 4), JY25 (the wild-type control) (lane 5), white blood cells (WBCs) from J20 (lane 6), WBCs of a healthy volunteer (lane 7), or no DNA template (negative control, lane 8) was used for PCR analysis using primer sets designed according to the flanking regions of BP1, BP2, and BP3 (illustrated in Figure 1). Both the integrity and quantity of the DNA templates were confirmed by PCR using control primers (labeled Control). That appropriate-sized PCR products were amplified using DNA derived from the circulating WBCs of J20 (lane 6) shows that both the S1 and L1 versions of chromosome 12 were present in vivo. (B) Confirmation of the breakpoints in US1 by PCR. Genomic DNA from WBCs of a healthy volunteer (lane 1), bone marrow cells of US1 (lane 2), PMN of US1 (lane 3), US1W (the hybrid cell line containing wild-type chromosome 12) (lane 4), or US1M [the hybrid cell line containing (der(12)] (lane 5), or no DNA template (negative control, lane 6) were used for PCR analysis using primer sets designed according to the sequence of flanking regions of the BP5, BP6, BP7, and BP8 (illustrated in Figure 1). These experiments show that both wild-type chromosome 12 and der(12) were present in the peripheral blood and bone marrow of US1. (C) Metaphase FISH showing the chromosomal abnormality in J20. BAC probes 471G7, 150C16, and 366L20 were hybridized against chromosomal specimens derived from the cell line (L1) that contains only long chromosome 12. Two hybridization signals (arrows) were detected with all 3 BAC probes, confirming that long chromosome 12 contained the inserted material deleted from short chromosome 12. (D) Metaphase FISH showing the chromosomal abnormality in US1. BAC probes 366L20, 438I19, and 474P2 (illustrated in Figure 1) were hybridized with chromosomal specimens derived from bone marrow cells of US1. Each sample contained a der(12) (indicated by 2 hybridization signals on the same chromosome, double arrows) and a wild-type chromosome 12 (indicated by one hybridization signal, arrow). The intrachromosomal insertion splits the signal on 12p generated by hybridization of probe 438l19, whereas signals are generated on 12q and 12p when probes that overlap BP5 on the centromeric (474P2) and telomeric (366L20) ends are used.