Principles of the Hpa II- McrBC whole-genome-amplification PCR (HM-WGA-PCR) method. The two parallel lines in the "Digestion" panel indicate genomic amplicons from both alleles. The circled "m" indicates a methylated CpG dinucleotide. Hpa II digests unmethylated CCGG, but not methylated CmCGG. In contrast, McrBC digests methylated RmC40-80RmC sequences, but not unmethylated RC40-80RC. Genomic DNA is digested with Hpa II and McrBC independently. Subsequently, an aliquot of each restriction-enzyme-digested DNA (50 ng) is subjected to whole-genome-amplification (WGA) to yield 5 μg of whole-genome-amplified DNA. Using an aliquot of the amplified DNA (50 ng), the target DNA region is PCR-amplified by the primer pair (dotted arrows). The PCR products from the Hpa II/Hha I-digested and McrBC-digested DNA are electrophoresed, stained with ethidium bromide, and visualized by UV illumination. If an amplicon is fully methylated (i.e., complete methylation), it is digested by McrBC, but not by Hpa II. Thus, it is amplified from the Hpa II-digested and whole-genome-amplified DNA, but not from the McrBC-digested and whole-genome-amplified DNA. By contrast, if an amplicon totally escapes methylation (i.e., null methylation), it is digested by Hpa II, but not by McrBC. Thus, it is amplified from McrBC-digested and whole-genome-amplified DNA, but not from Hpa II-digested and whole-genome-amplified DNA. If an amplicon contains both methylated and unmethylated alleles (i.e., composite methylation), it is amplified from both whole-genome-amplified templates. If an amplicon is partially methylated on both alleles (i.e., incomplete methylation), it is amplified from neither whole-genome-amplified template.