text: Methylation-specific restriction-PCR-assay
  DNA was extracted with a commercial kit (Qiagen) as recommended. Since the restriction ability of several endonucleases is inhibited by methylation of their target sequence, we used methylation-sensitive enzymes HpaII and HaeII-isochizomer Bsp143II and Bsh1236I (MBI Fermentas, St Leon-Rot, Germany) (20,24). As control the methylation-resistant enzyme MspI and an enzyme with no recognition site in the target promoter, EcoRI, were used. DNA (0.8 microg) was digested by 40 U the respective enzyme for 6 h and, to ensure complete cleavage, additional 20 U for 16 h. Thereafter 100 ng of digested DNA was used to a PCR amplification of two fragments (F1 and F2) spanning part of the IRF-4 promoter (30) (GenBank U52683; see Figure 3A). The sequences of the primers were F1-forward: 5'-TTGAGATGGAGTCTTGCTCTGT-3', F1-reverse: ATCACTTCCAGACTTCAGTTCACCT-3' (341 bp); F2-forward: 5'-AAGGTGAACTGAAGTCTGGAAGTGA-3', F2-reverse: 5'-CCAGGACCTCAGGAGGCCAGTCA-3' (474 bp). The PCR conditions were described elsewhere (3). PCR was performed with an annealing temperature of 62degreesC and 35 cycles. When DNA was methylated at specific sites, the sensitive enzymes were not able to digest the DNA and amplification took place; in case of no methylation, DNA was digested and no product was generated. The PCR products were electrophoresed on a 3% agarose gel, were stained with ethidium bromide and photographed. PCR products were verified by automated sequencing. 
  