Inverse PCR

For the detection of single chromosomal translocations an adaptation of an inverse PCR protocol³⁸ was performed. After treatment, genomic DNA was extracted with the DNeasy Blood and Tissue Kit (Qiagen). 10 µg of DNA were incubated for 1 h with Shrimp Alkaline Phosphatase (NEB). After inactivation and digestion with either Bgl2 or Sac1 (NEB) DNA was purified with QIAquick PCR Purification Kit (Qiagen). To ensure ligation in cis of DNA molecules DNA was diluted at 18 µg/ml and ligated for 20 h at 4 °C. After purification and quantification 40ng of DNA were used per PCR reaction. Two consecutive PCR reactions (LongAmp polymerase, NEB) were performed to ensure specific amplification. First-round primers: MLLBgl2_1: (5′-GAGGAAATCAGCACCAACTGGGGGAA-3′) and MLLBgl2_2: (5′-GATCCTGTGGACTCCATCTGCTGGAA-3′) or MLLSac1_1 (5′-ATATGGGTGCAAAGCACTGTAT-3′) and MLLSac1_2 (5′-ACCAGTCCTTCAACTTCTGGG-3′). One microliter of a 1 : 10 dilution of the first-round PCR mix was amplified with second-round primers: MLLBgl2_3: (5′-CATTAGCAGGTGGGTTTAGCGCTGGG-3′) and MLLBgl2_4: (5′-TTCTCCTGCTTATTGACCGGAGGTGG-3′) or MLLSac1_3 (5′-CAGTAGACCCCTGGCACTTG-3′) and MLLSac1_4 (5′-AGCAATCTCACAGGGTTCCT-3′). Novel-size bands were purified from agarose gel and sequenced using MLLBgl2_4 or MLLSac1_5 (5′-AGTGCAGTGGCGTGATAATG-3′) primers. Translocations were represented as by a Circos plot³⁹ using the OmicCircos package of R.