7 deaza dgtp

Telomere fusions were determined using TAR-fusion PCR assay [50] (link) with minor modifications. Two-step touchdown PCR was performed in a 20 μl reaction mixture using 50 ng of DNA, multiple primers, 10% 7-Deaza-dGTP (Roche Diagnostics), and Advantage GC Genomic LA Polymerase Mix (Clontech Laboratories). Multiple PCR primers were designed within TAR1 (Telomere Associated Repeat 1)–like sequences common to many chromosomes distal regions [56] (link). The primer sequence information and PCR condition are shown in Tanaka et al. [50] (link). As an internal positive PCR control, the following primers were used for amplifying ancestral interstitial telomere head-to-head fusion within chromosome 2q13-q14.1 [57] (link): 5′-GCA AGG CGA GGG GCT GCA TTG CAG GGT GAG-3′, 5′-CAG CAG GGG GCG CTG GAC AGC ACT GTA AG-3′. TAR fusion PCR products were dot-blotted onto Hybond⁺ membranes and hybridized using a DIG-labeled telomere probe (Roche Diagnostics). Six PCR reactions per each case were used for every PCR. Telomere fusion was defined as ‘negative’ when TAR-fusion PCR did not amplify any products using total twenty PCR reactions which correspond to more than 1 × 10⁵ cells.