Ligation-mediated PCR for DNA break detection

Supernatant was removed from agarose plugs, replaced with 50 μl fresh 1× ligase buffer, and plugs were heated to 62°C to melt agarose. 20 μl DNA (∼10,000 cell equivalents) was added to 2 μl T4 DNA ligase (2 Weiss units, MBI Fermentis), 10 μl ds annealed linker in 1× ligase buffer, 3 μl 10× ligase buffer, and 30 μl dH₂0, and incubated overnight at 18°C. Linker was prepared by annealing 5 nmol each of LMPCR.1 (5′-GCGGTGACCCGGGAGATCTGAATTC-3′) and LMPCR.2 (5′-GAATTCAGATC-3′) in 300 μl 1× ligase buffer, which results in a ds oligo with a 14-nt ss overhang that can only ligate unidirectionally. Ligated DNA samples were heated to 70°C for 10 min, diluted fivefold in distilled H₂0, and assayed for GAPDH by PCR to adjust DNA input before LM-PCR. The following primers (Integrated DNA Technologies) were used in conjunction with linker primer (LMPCR.1) to amplify DNA breaks: 5′Sμ-GCAGAAAATTTAGATAAAATGGATACCTCAGTGG-3′ (used for all experiments except for data shown in Fig. S1); 3′Sμ: 5′-GCTCATCCCGAACCATCTCAACCAGG-3′ (used only for Fig. S1); Sg3-AP: 5′-AACATTTCCAGGGACCCCGGAGGAG-3′ (25 (link)); and CmuL2: 5′-CTGCGAGAGCCCCCTGTCTGATAAG-3′ (42 (link)).
Three-fold dilutions of input DNA (0.5, 1.5, and 4.5 μL) were amplified by Hotstar Taq (QIAGEN) using a touchdown PCR program. PCR products were run on 1.25% agarose gels and vacuum blotted (VacuGene XL, Pharmacia) onto nylon membranes (GeneScreen Plus, PerkinElmer). Blots were hybridized with oligonucleotide probes end-labeled with γ³²P-ATP at 37°C overnight and washed at 55°C with 2X SSC/0.1%SDS. The following probes were used: μ probe 5′: 5′-AGGGACCCAGGCTAAGAAGGCAAT-3′; Sμ probe: 5′-GTTGAGAGCCCTAGTAAGCGAGGCTCTAAAAAGCACGCT-3′ (7 (link)); Sμ3′ probe: 5′-GGGCTGGCTGATGGGATGCCCC-3′ (used only for Fig. S1); Sγ3-LP: 5′-GGACCCCGGAGGAGTTTCCATGATCCTGGG-3′ (25 (link)); and Cμ: 5′-TGGCCATGGGCTGCCTAGCCCGGGACTTCCTG-3′ (42 (link)).