Schematic of methylation profiling technique. DNA is harvested from samples (1) and digested with methylation-sensitive enzyme, Hpa II, which cuts at its CCGG target site only if the CpG is not methylated (2). The DNA is then size-fractionated on a sucrose gradient, followed by determination of fragment size by agarose gel electrophoresis in order to choose fractions containing fragments > approximately 80 bp and < approximately 2.5 kb; in this example, fractions represented by lanes numbered 3 to 10 (3). Test and reference samples are then differentially labeled (4) and competitively hybridized to a clone-based microarray containing 2,049 clones for chromosome 1, 17 clones for the X chromosome, and 70 other clones (5). The signal intensities of each fluorescence channel are expressed as a ratio, log2 transformed, and then loess normalized on the percent GC content of clone sequences (6).