Fig. 1

Flowchart of MSD-library preparation. Genomic DNA (100 ng) was digested with 10 units of the primary restriction enzyme SbfI for 1 h and then ligated with 0.5 nmol Adaptor A using 400 units of T4 DNA ligase for 2 h. The treated sample was then digested with 100 units of the non-methylation-sensitive restriction enzyme MspI (100 units) followed by ligation of the ends of the DNA fragment with Adaptor B. The ligated DNA fragments were then digested with 50 units of HpaII for 1 h. Owing to the methylation sensitivity of HapII, only DNA fragments with a methylated CpG retained Adaptor B, which was removed from all other fragments. The DNA fragments were then subjected to Pre-PCR using specific primers for Adaptor A and Adaptor B. Fragments that did not contain Adaptor B at this stage were not amplified. The Pre-PCR amplicons (MSD library) were then amplified as a subpopulation by selective-PCR with 6-carboxyfluorescein (6-FAM)-labelled selective-PCR primers. Finally, the selective-PCR products were electrophoresed with a capillary sequencer and separated by length