Fig. 1

Flowchart of sample preparation for the amplification of methyl-CpG DNA fragments and labeling. The first restriction enzyme digestion is performed with Nco I (alternatively, other 5′-, or 3′-overhanging sticky-end restriction enzymes can be used) and HinP1 I (alternatively, other methyl-sensitive sticky-end restriction enzymes, such as Hha I, Hpa II can be used). If the B-HinP1 I site is unmethylated and the B′-HinP1 I site is methylated, the resulting Fragment-AB will be amplified in by the first PCR after the first ligation with Adaptor-1 and Adaptor-2. By the second digestion of the first PCR product with restriction enzyme HinP1 I, the amplified the Fragment-AB will be cut to form the smaller Fragment-B’B. After ligation with Adaptor-3, the Fragment-B’B will be amplified in the second PCR, which means that the B′-HinP1 I site is methylated in genomic DNA. In the case of the B′-unmethylated state, the Fragment-AB’ is amplified in the first PCR, but the Fragment-B’B is not amplified in the second PCR at all