Typhoon 9400 phosphoimager

The method employed for the assay probing for C-circles was slightly modified from that performed in Henson et al.^{19 (link)}. Briefly, genomic DNA was prepared as above. Digested DNA was cleaned up by phenol-chloroform extraction and precipitation. DNA was diluted and measured using a Nanodrop spectrophotometer. Generally, 100 and 50 ng of DNA were used for each sample (10 μl). DNA was combined with 10 μl 0.2 mg/ml BSA (NEB), 0.1% Tween, 1 mM each dATP, dGTP and dTTP, 1X ϕ29 Buffer (NEB) and 7.5 U ϕ29 DNA polymerase (NEB) and incubated at 30 °C for 12 h then 65 °C for 20 min. Reaction products were diluted to 100 μl with 2XSSC and dot-blotted onto a 2XSSC soaked nylon membrane. DNA was UV-cross-linked onto the membrane, and then hybridized at 50°C with end-labeled ³²P-(CCCTAA)₄ oligo probe. Blots were washed, exposed and scanned using a Typhoon 9400 PhosphoImager (Amersham, GE Healthcare) and analyzed using ImageQuant Software.

Telomere gels were performed using telomere restriction fragment (TRF) analysis. Genomic DNA was digested using AluI and MboI (NEB). 5 μg of DNA was run on a 1% PFGE agarose gel (Bio-Rad) in 0.5 × TBE buffer using the CHEF-DRII system (Bio-Rad) at 6V cm⁻¹; initial switch time 1 s, final switch time 6 s, for 17hrs at 14°C. The gel was then dried for 2hrs at 60°C, denatured in a 0.5N NaOH 1.5M NaCl solution, and neutralized. The gel was hybridized with ³²P-labeled (TTAGGG)₄ oligonucleotides in Church buffer overnight at 55°C. The next day, the membrane was washed three times in 2 × SSC buffer and once in 2x SSC 0.5% SDS, exposed onto a storage phosphor screen and scanned using Amershan™ Typhoon (GE Healthcare). Telomere length was determined using TeloTool software. To detect TTAGGG and TCAGGG variants from genomic DNA using a Dot Blot system, 2 μg of AluI and MboI (NEB) digested genomic DNA was diluted to 100 μL with 2 × SSC, heated at 95°C for 5mins, cooled on ice and dot-blotted onto a 2 × SSC-soaked nylon membrane. DNA was UV cross-linked onto the membrane and hybridized with P³² end-labeled oligos (CCCTAA)₄ or (TCAGGG)₄ products. An Alu probe was used as a loading control. Blots were denatured, neutralized, washed, exposed to PhosphoImager screens, scanned using a Typhoon 9400 PhosphoImager (GE Healthcare) and quantified with ImageJ.

Telomere gels were performed using telomere restriction fragment (TRF) analysis. Genomic DNA was digested using AluI and MboI (NEB). Then, 4–10 μg of DNA was run on a 1% PFGE agarose gel (Bio-Rad) in 0.5× TBE buffer using the CHEF-DRII system (Bio-Rad) at 6 V cm⁻¹; the initial switch time was 1 s, and the final switch time 6 s, for 17 h at 14 °C. The gel was then dried for 2 h at 60 °C, denatured in a 0.5 M NaOH/1.5 M NaCl solution, and neutralized. Gel was hybridized with ³²P-labeled (TTAGGG)₄ oligonucleotides in Church buffer overnight at 55 °C. The next day, the membrane was washed three times in 2× SSC buffer and once in 2× SSC 0.5% SDS, exposed onto a storage phosphor screen and scanned using Typhoon 9400 PhosphoImager (GE Healthcare). Telomere length was determined using TeloTool software.

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.