Definition of the boundaries of the deletion on chromosome 2 present in the cell line STA-NB-10/dmin using FISH. (a,b) Partial metaphases showing examples of FISH experiments using fosmid probes spanning the distal (a) and proximal (b) breakpoints of the deleted region, cohybridized with the BAC containing the MYCN gene. (c,d) The position on the human sequence of fosmid clones in a and b, respectively. (Vertical red lines) Amplicon boundaries that were defined by sequencing the amplicon junction (see text and Supplemental Fig. 4). The definition of the deletion breakpoints was obtained as follows. Distal breakpoint. Fosmid G248P87145E2 (chr2:15,461,044–15,505,473; 44.4 kb) gave a signal on del(2) that, by eye, consistently appeared to be half the intensity of the signal on normal chromosome 2 (10 analyzed metaphases; its black and white original signal is separately reported in a). The middle part of the fosmid was therefore assumed to harbor the telomeric break of the deletion at ∼chr2:15,483 kb. G248P87145E2 is present also on the amplicon (a, inset), but the signal intensity is biased by the amplification. The normal intensity on del(2) of fosmid G248P800648F12 (chr2:15,428,940–15,471,782), and the absence of any signal on the dmin, was consistent with the assumption in a. Proximal breakpoint. Fosmid G248P87325H8 (chr2:18,656,707–18,695,100; 38.4 kb) consistently gave a signal of half intensity on del(2) (10 analyzed metaphases; its black and white original signal is separately reported in b). The centromeric break of the deletion was therefore assumed to lay at ∼chr2:18,575 kb. Note that sequences of G248P87145E2 are present on the amplicon (b, inset), but, again, the signal intensity is biased by the amplification. The intensity on del(2) of signals yielded by fosmids G248P89303A7, G248P85351E4, and G248P86949C3 was consistent with this hypothesis. The deletion, therefore, spanned approximately the interval chr2:15,483–18,575 kb. Because of growth difficulty of the STA-NB-10/hsr cell line, not all FISH experiments performed on STA-NB-10/dmin were also performed in hsr subclone (see Supplemental Table 1). (e,f) Evidence for the deletion on one of the two copies of chromosome 2. Two fosmid clones flanking the deleted region (red and green) were hybridized with a probe (RP11-723P4, violet) mapping within the deletion but absent in the amplicon (see text). The BAC was chosen in order to avoid the high background due to the amplification. The distance between the red and green probes on normal chromosome 2 (red arrow) is substantially reduced on the del(2) (white arrow), as expected. (f) Rationale of the FISH experiment in e.
