Experimental design and chromosomal mapping of variably methylated targets. (a) Experimental design of methylation analysis with Affymetrix human promoter 1.0R microarray. In this adaptor-mediated, PCR-based approach to probe the microarrays, (i) DNA was digested with MseI, purified and then (ii) adaptors were ligated before (iii) a second digestion was performed with the methylation-sensitive HhaI enzyme. (iv) Samples were then PCR amplified and (v) these PCR products were fragmented, labeled, and hybridized to the Affymetrix Human Promoter 1.0R GeneChips. Open circles, unmethylated cytosines; filled circles, methylated cytosines. (b) The chromosomal location of variably methylated promoter array gene targets in the MDA-MB-468GFP-LN cells relative to the parental cell line are shown as red blocks and represent significant (P < 0.05) targets identified in at least two of three replicates.