Fig. 5

Nested IPCR detection of DNA breakages within the ABL gene in H₂O₂-treated HK1. HK1 cells were seeded in 60-mm culture dishes and were grown to optimal density (60–70% confluency). The cells were then either untreated (lane 3) or treated with 1 μM (lanes 4, 7, 10 and 13), 10 μM (lanes 5, 8, 11 and 14) or 50 μM (lanes 6, 9, 12 and 15) of H₂O₂ for 2 h (lanes 4–6), 4 h (lanes 7–9), 6 h (lanes 10–12) and 8 h (lanes 13–15). Genomic DNA was isolated and manipulated for nested IPCR. In the modification for nested IPCR, the DNA samples were either subjected to digestion with Age I (a) or double digestion with Age I and EcoR I (b). The IPCR products were analysed on 1% agarose gel. Side arrow in panel a indicates the position of the 3 kb IPCR bands resulting from the amplification of the intact ABL gene. Side brackets in both panels a and b indicate the possible IPCR bands from the ABL cleaved fragments. Negative control for PCR was included (lane 16). This IPCR result is representative of 2 repeats with similar results. M₁: 1 kb DNA ladder. M₂: 100 bp DNA ladder