Amershan typhoon

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.