φdna polymerase

CC assay was performed as described (Henson et al., 2009 (link)). Genomic DNA was purified, digested with AluI and MboI and cleaned up by phenol-chloroform extraction and precipitation. DNA was diluted in ultraclean water and concentrations were exhaustively measured to the indicated quantity (generally, 30, 15 and 7.5ng) using a Nanodrop (ThermoFisher). Samples (10 μl) were combined with 10μl BSA (NEB; 0.2 mg/ml), 0.1 % Tween, 0.2 mM each dATP, dGTP, dTTP and 1^χ Φ29 Buffer (NEB) in the presence or absence of 7.5U ΦDNA polymerase (NEB). Samples were incubated at 30°C for 8hrs and then at 65°C for 20mins. Reaction products were diluted to 100μl with 2^XSSC and dot-blotted onto a 2^xSSC-soaked nylon membrane. DNA was UV cross-linked onto the membrane and hybridized with a P₃₂ end-labelled (CCCTAA)₄ oligo probe to detect C-circle amplification products. All blots were washed, exposed to Phospholmager screens, scanned using a Typhoon 9400 Phospholmager (GE Healthcare) and quantified with Image J. In all reactions, when Φ29 was omitted as a negative control DNA was used.

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.