(A) Single-step methylation-specific PCR is shown for theMSH6 locus. Lanes 1, 3, 4, 6, and 7: samples from patients in acute-phase CML; lanes 2 and 5: samples from patients in chronic phase CML; lane 8: normal donor buffy-coat DNA; lane 9: normal donor buffy-coat DNA treated in vitro with CpG methylase (SssI methylase). M, primers specific for methylated DNA; U, primers specific for unmethylated DNA; ◊x, ◊x/HaeIII marker. (B) Two-step methylation-specific PCR for the ATM promoter region. In the first round, the CpG island is amplified from bisulfite-modified DNA using primers that are insensitive to methylation (see Materials and Methods). In the second round, primers specific for methylated DNA (M) or unmethylated DNA (U) are used in PCR together with a small amount of template from the first reaction (arrow). Lane 1: normal donor buffy-coat DNA; lane 2: normal donor buffy-coat DNA treated in vitro with CpG methylase (SssI methylase); lanes 3 through 6: samples from patients in acute-phase CML. ◊x, ◊x/HaeIII marker.