Telomeric DNA Detection Assays

Both assays have been performed as described previously^{21 (link)}. Briefly, genomic DNA was purified and digested with AluI and MboI. For restriction-fragment analysis, 10 µg of digested DNA was electrophoresed on a 0.8% TBE-agarose gel. Subsequently, telomeric DNA was detected by Southern blotting using a [³²P]dATP end-labelled (CCCTAA)₄ oligonucleotide probe. For the C-circle assay, DNA samples (7.5 ng, 10 µl) diluted in ultraclean water were combined with 10 µl BSA (NEB; 0.2 mg ml⁻¹), 0.1% Tween, 0.2 mM each dATP, dGTP, dTTP, and 1 × Φ29 Buffer (NEB) in the presence or absence of 7.5 U ΦDNA polymerase (NEB), incubated at 30 °C for 8 h and then at 65 °C for 20 min. Reaction products were diluted to 100 µl with 2 × SSC and dot-blotted onto a 2 × SSC-soaked nylon membrane. DNA was ultraviolet cross-linked onto the membrane and hybridized with a ³²P-end-labelled (CCCTAA)₄ oligonucleotide probe to detect C-circle amplification products. All blots were washed, exposed to PhosphoImager screens, scanned, and quantified using a Typhoon 9400 PhosphoImager (Amersham, GE Healthcare). Genomic DNA from ALT-positive (U2OS) cells served as positive control and reference for the quantification of C-circles detected in other cell lines.